Bioprotocols

shRNA Methods

2011-06-30T22:08:00.000-07:00

shRNA Protocol
Silencing Plasmids into mammalian cells. Amounts listed above are based on use of 6-well plates. shRNA Transfection Protocol. Santa Cruz Biotechnology, Inc.
datasheets.scbt.com/shRNA_protocol.pdf

Access : Efficient transfection of DNA or shRNA vectors into ...
29 Nov 2007 ... We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat ...
www.nature.com

QIAGEN - Attractene Transfection Reagent - For efficient DNA ...

Transfection of shRNA (short-hairpin RNA) vectors for gene silencing ... and plate format to receive a QIAGEN transfection protocol to print out or download ...
www.qiagen.com/.../attractenetransfectionreagent.aspx

shrna transfection Protocol recipe

Results 1 - 8 of 12 for shrna transfection Protocol recipe. (0.03 seconds) ... This protocol is focused on the use of shRNAs in the widely . ...
www.vadlo.com/.../q?...shrna%2Btransfection%2BProtocol%2Brecipe...

RNAi Transfection Protocols

Invitrogen has the most complete collection of transfection reagents with exceptional ...Human Mesenchymal Stem Cells, Human bone marrow, Protocol ...
www.invitrogen.com

Ambion TechNotes 12(1): Optimizing siRNA Transfection for RNAi

Once a protocol is optimized for a particular cell type, reproducible siRNA screening experiments can easily be done. Efficient Transfection is Critical ...
www.ambion.com

Infection of gene specific shRnA into cells via retrovirus ...
Transfect the shRNA plasmids** into the packaging cells by fol- ... tRAnsfectIon. Transfect the cells using the protocol above for transient transfection. ...
www.origene.com/.../HuSH_29_retroviral__infection_GeneDex.pdf

Chemical recipe in shrna transfection Protocol

Results 1 - 8 of 15 for Chemical recipe in shrna transfection Protocol. (0.04 seconds) ... This protocol is focused on the use of shRNAs in the widely . ...
www.vadlo.com/.../q?...Chemical%2Brecipe%2Bin%2Bshrna%2Btransfection%2BProtoco...

siRNA Transfection - Protocols, techniques, methods

... pre-optimized transfection reagents and siRNA services, shRNA-expressing DNAtransfection, microRNA, In Vivo siRNA transfection systems, high-throughput ...
www.sirnatransfection.org

shRNA Protocols

27 Nov 2008 ... Lentiviral Transduction Protocol.doc (Sigma). Nature Protocols: Efficienttransfection of DNA or shRNA vectors. ...
www.e-biotek.com/rna/227-shrna-protocols.html

shRNA transfection

15 Jul 2009 ... Maximize shRNA delivery with Arrest-In transfection reagent. Arrest-In™transfection reagent from Open Biosystems is a polymeric ...
www.gentaur.com/shrna_transfection.htm

shRNA GFP protocol
OZ Biosciences / Protocol shRNA GFP vs. 1.0 / www.ozbiosciences.com / ... (such asshRNA Luciferase) and efficient transfection reagents such as Lullaby or ...
www.ozbiosciences.com/docman/sirna...protocol/download-2.html

Efficient transfection of DNA or shRNA vectors into neurons using ...

14 Dec 2010 ... We provide a protocol for transfection of cDNA and RNA interference (shRNA) vectors, using magnetofection, into rat hippocampal neurons ...
www.zora.uzh.ch/138/

shRNA & miRNA Plasmid Transfection

Efficient gene silencing can be obtained with small hairpin RNA (shRNA) or ... jetPRIME™ convenient protocol for DNA, siRNA and co-transfection of DNA and ...
www.polyplus-transfection.com/transfection.../shrna-mirna-plasmid-transfection-jetprime/

Design and Cloning of an shRNA into a Lentiviral Silencing Vector ...

1 Aug 2008 ... Certain brands of FBS do not support efficient transfection and can result in ....Related Protocol. Design and Cloning of an shRNA into a ...
cshprotocols.cshlp.org/cgi/content/full/2008/8/pdb.prot5009

Methods to determine the size of an object in microns

2011-06-30T22:05:00.002-07:00

Mathematically

Th number of pixels the image of a cell takes on the CCD camera depends on:

1. the magnification used, and on

2. the physical size of the pixels on the CCD camera (in our case, it is 6.45um)

In order to relate the size of a cell in pixels to its size in um, use the following formula:

Cell size (per pixel) = Physical length of a pixel on the CCD / total magnification

The physical length of a pixel on our CCD is 6.45um. So for the following magnification, this formula gives us:

l 100x : 0.0645 um/pixel or 15.50 pixels/um

l 90x : 0.0717 um/pixel or 13.95 pixels/um

l 60x : 0.1075 um/pixel or 9.30 pixels/um

l 40x : 0.1613 um/pixel or 6.21 pixels/um

l 20x : 0.3225 um/pixel or 3.10 pixels/um

l 10x : 0.645 um/pixel or 1.55 pixel/um

Important notes:

l We assumed here that the magnifier is at position 1x (not 1.5x). If it is at 1.5x, total magnification must be multiplied by 1.5.

l We assumed here that the bin size is 1x1. If it is, say, 2x2, the size of a pixel will be double.

l 90x corresponds to 60x objective with 1.5x intermediate magnification

Calibration slide

Use a calibration slide which has a grid with known line-to-line spacing (9.9um). Align the slide so that the grid lines are parallel/perpendicular to the x and y axes. I borrowed such a slide from Peter Sorger's lab.

l 60x DIC Plan Apo

1. 13 squares gave 1204.26 pixels in length. 1204.26 pixels/(13 square * 9.9um/squares) = 9.35 pixels/um

2. 14 squares gave 1297.30 pixels in length. 1297.30 pixels/(14 squares * 9.9um/squares) = 9.36 pixels/um

l 60x DIC Plan Apo with 1.5x intermediate magnification:

1. Horizontal : 3 measurements of 8 squares gave 1106.2, 1105.2 and 1106.2 pixels in length. --> 13.96' pixels/um (Std error = 0.00)

2. Vertical: 3 measurements of 7 squares gave 968.2, 968.2, 968.2 pixels in length --> 13.97 pixels/um (Std error = 0.00)

3. 60x Ph Plan Apo with 1.5x intermediate magnification gave virtually the same results.

xy-motorized stage

Put a sample on a slide or pad (cells, sphere) or find a grain of dust on the slide or pad. Record the position of the sample. Use IPLab to tell the stage to move a certain distance in um. Determine the distance (in pixels) between the sample's former position and its new position.

l 60x

*Based on three samples I got 9.22 +/- 0.13 pixels/um (error represents the standard deviation)

To test the calculated um/pixel for a given objective, one could do the following: Get the calibration slide. Take a picture. Knowing how wide (in pixels) our CCD camera is, determine the width (in um) of the CCD image. Move the stage by that distance. Take another picture. Put the two pictures next to one-another. Do the grids line-up?

(source: openwetware)

Histological Fixatives and Sample Fixation

2011-06-30T22:05:00.001-07:00

Acetic alcohol - fixation time 1 minute.

(For smears, cytospin preparations or frozen sections).

95% methanol - 100ml

Glacial acetic acid - 3ml.

The sections should be washed in water before staining.

Bouin's fluid - fixation time 6 hours.

Saturated aqueous solution of picric acid - 75ml

Formalin ( - 40% aqueous solution of formaldehyde) - 25ml

Glacial acetic acid - 5ml

Fixed tissue should be transferred to 70% alcohol.

Carnoy's fluid - fixation time 1-3 hours.

Ethanol - 60ml

Chloroform - 30ml

Glacial acetic acid - 10ml

Fixed tissue should be processed immediately or transferred to 80% alcohol.

Formol sublimate - fixation time 4-6 hours.

Formalin (40% aqueous solution of formaldehyde) - 100ml

Mercuric chloride (saturated aqueous) - 900ml

Fixed tissue should be transferred to 80% alcohol.

Helly's fluid - fixation time 12-24 hours.

Stock solution:

Potassium dichromate - 25g

Mercuric chloride - 50g

Sodium sulphate - 10g

Distilled water - 1000ml

For use:

Stock solution - 100ml

Formalin ( - 40% aqueous solution of formaldehyde) - 5ml

The fixative solution should be made up just before use. Fixed tissue must be washed for 24 hours in running tap water prior to
processing.

Neutral buffered formalin - fixation time 12-24 hours.

Formalin ( - 40% aqueous solution of formaldehyde) - 100ml

Sodium dihydrogen orthophosphate (monohydrate) - 4g

Disodium hydrogen orthophosphate (anhydrous) - 6.5g

Distilled water - 900ml

This fixative is suitable for most histological purposes. It is to be preferred to formol-saline (a single 10% solution of formalin in
0.9% aqueous NaCl) as formalin pigment is avoided. Specimens may be stored in this fluid. The solution is isotonic.

Michel's fixative for immunoflourescence - fixation time 24-48 hours.

Buffer :

0.81g potassium citrate

0.0625g N-ethylmaleimide - HANDLE WITH CARE!

0.123g magnesium sulphate

100mls distilled water

Before use add 55g ammonium sulphate and allow to dissolve.

Adjust pH to 7.0-7.2 with 1M KOH.

Place tissue biopsies in fixative for 24-48 hours. Wash tissues in buffer, three times over 10 minutes, and freeze at -70C.

Paraformaldehyde - fixation time depends on technique to follow.

Sodium dihydrogen orthophosphate 2.26% - 41.5ml

Sodium hydroxide 2.52% - 8.5ml

Heat to 60-80C in a covered container.

Add 2g `Analar' paraformaldehyde and stir until dissolved. Filter.

Zenker's fluid - fixation time 4-24 hours.

Distilled water - 950ml

Potassium dichromate - 25g

Mercuric chloride - 50g

Glacial acetic acid - 50g

Fixed tissue should be washed overnight in running tap water before processing.

REMOVAL OF FIXATION Pigments

Formalin pigment

1. Dewax the sections, rinse in 100% alcohol, rinse in 70% alcohol, rinse in distilled water.

2. Treat in saturated alcoholic picric acid for 30 minutes to 2 hours.

3. Wash well in running tap water.

4. If yellow staining of the section persists rinse in dilute lithium carbonate.

5. Rinse in tap water.

6. Continue with method.

Mercury pigment

1. Dewax the sections, rinse in 100% alcohol, rinse in 70% alcohol, rinse in distilled water.

2. Treat in Lugols iodine for 2 minutes.

3. Decolourise in 5% sodium thiosulphate for 5 minutes.

4. Wash well in running tap water.

5. Continue with method.

Dichromate pigment

1. Dewax the sections, rinse in 100% alcohol, rinse in 70% alcohol, rinse in distilled water.

2. Treat in 2% HCl in 70% alcohol 16-24 hours.

3. Rinse in tap water.

4. Continue with method.

Citrate test

2011-06-30T22:04:00.000-07:00

Principle

The citrate test detects the ability of an organism to use citrate as the sole source of carbon and energy. Bacteria are inoculated on a medium containing sodium citrate and a pH indicator such as bromothymol blue. The medium also contains inorganic ammonium salts, which are utilized as sole source of nitrogen. Use of citrate involves the enzyme citritase, which breaks down citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and carbon dioxide (CO2). Production of sodium bicarbonate (Na2CO3) as well as ammonia (NH3) from the use of sodium citrate and ammonium salts results in alkaline pH. This results in a change of the medium’s color from green to blue.

Bacterial colonies are picked up from a straight wire and inoculated into slope of Simmons citrate agar and incubated overnight at 37 °C. If the organism has the ability to use citrate, the medium changes its color from green to blue.

Examples:

Escherichia coli: Negative

Klebsiella pneumoniae: Positive

Frateuria Aurantia: Positive

Method

Inoculate Simmons citrate agar (do not inoculate heavily) by using straight wire from an 18 to 24 hour old colony. Inoculating from a broth culture is not recommended because the inoculum would be too heavy. Incubate at 35°C for up to seven days.

Results

Positive : Growth on the medium even without colour change will be considered as positive. A colour change in the medium would be observed if the test organism produces acid or alkali during its growth. The usual colour change observed is from green (neutral) to blue (alkaline).

Negative : No growth observed.

NCBI: Training & Tutorials

2011-06-30T22:03:00.000-07:00

NCBI：National Center for Biotechnology Information

U.S. government-funded national resource for molecular biology information. Access to many public databases and other references, ...
www.ncbi.nlm.nih.gov/

DATABASES

NCBI C++ Toolkit Manual

A comprehensive manual on the NCBI C++ toolkit, including its design and development framework, a C++ library reference, software examples and demos, FAQs and release notes. The manual is searchable online and can be downloaded as a series of PDF documents.

NCBI Education Page

Provides links to tutorials and training materials, including PowerPoint slides and print handouts.

NCBI Glossary

Part of the NCBI Handbook, this glossary contains descriptions of NCBI tools and acronyms, bioinformatics terms and data representation formats.

NCBI Handbook

An extensive collection of articles about NCBI databases and software. Designed for a novice user, each article presents a general overview of the resource and its design, along with tips for searching and using available analysis tools. All articles can be searched online and downloaded in PDF format; the handbook can be accessed through the NCBI Bookshelf.

NCBI Help Manual

Accessed through the NCBI Bookshelf, the Help Manual contains documentation for many NCBI resources, including PubMed, PubMed Central, the Entrez system, Gene, SNP and LinkOut. All chapters can be downloaded in PDF format.

NCBI Website Search

A database of static NCBI web pages, documentation, and online tools. These pages include such content as specialized online sequence analysis tools, back issues of newsletters, legacy resource description pages, sample code, and other miscellaneous resources. Searching this database is equivalent to a site search tool for the whole NCBI web site. FTP site is not covered.

DOWNLOADS

FTP: NCBI Field Guide Manual

Downloadable material for NCBI's previously offered Field Guide training course.

FTP: NCBI Structure Course Materials

PowerPoint slides, handouts and exercises for the previously offered NCBI course "Exploring 3D Molecular Structures."

NCBI Data Specifications

Specifications for NCBI data in ASN.1 or DTD format are available on the Index of data_specs page. The "NCBI_data_conversion.html" links to the conversion tool.

National Library of Medicine (NLM) DTDs

A suite of tag sets for authoring and archiving journal articles as well as transferring journal articles from publishers to archives and between archives. There are four tag sets: Archiving and Interchange Tag Set - Created to enable an archive to capture as many of the structural and semantic components of existing printed and tagged journal material as conveniently as possible; Journal Publishing Tag Set - Optimized for archives that wish to regularize and control their content, not to accept the sequence and arrangement presented to them by any particular publisher; Article Authoring Tag Set - Designed for authoring new journal articles; NCBI Book Tag Set - Written specifically to describe volumes for the NCBI online libraries.

TOOLS

Amino Acid Explorer

This tool allows users to explore the characteristics of amino acids by comparing their structural and chemical properties, predicting protein sequence changes caused by mutations, viewing common substitutions, and browsing the functions of given residues in conserved domains.

BLAST Tutorials and Guides

This page links to a number of BLAST-related tutorials and guides, including a selection guide for BLAST algorithms, descriptions of BLAST output formats, explanations of the parameters for stand-alone BLAST, directions for setting up stand-alone BLAST on local machines and using the BLAST URL API.

Coffee Break

Part of the NCBI Bookshelf, Coffee Break combines reports on recent biomedical discoveries with use of NCBI tools. Each report incorporates interactive tutorials that show how NCBI bioinformatics tools are used as a part of the research process.

E-Bench

This interactive tool allows users to build E-utility URLs, either from a form or by hand, and then view their raw output. The tool provides a simple environment for testing E-utility URLs before including them in applications.

Ebot

A tool that allows users to construct an E-utility analysis pipeline using an online form, and then generates a Perl script to execute the pipeline.

NCBI News

NCBI's monthly newsletter that provides information on new and updated databases, and software services. The News often has feature articles that highlight and demonstrate services, features, tools, and interesting data with practical examples of their use.

PSSM Viewer

Allows users to display, sort, subset and download position-specific score matrices (PSSMs) either from CDD records or from Position Specific Iterated (PSI)-BLAST protein searches. The tool also can align a query protein to the PSSM and highlight positions of high conservation.

PubMed Tutorials

A collection of web and flash tutorials on PubMed searching and linking, saving searches in MyNCBI, using MeSH and other PubMed services.

Science Primer

A basic introduction to the science and technology that underlies many of the NCBI resources. A great starting place for students and the general public, the Science Primer provides a basis for understanding the NCBI web site and mission, and provides direct links to many NCBI databases and tools. Topics include genome mapping, molecular modeling, mutations, microarrays (gene expression), genetics, pharmacogenomics (personalized medicine) and phylogenetics (evolutionary relationships).

H & E Staining Protocol

2011-06-30T22:02:00.002-07:00

Fixation of Tissues

1. Where the best possible morphology is required, animals should be anesthesized and subjected to cardiac perfusion with saline, followed by a 10% formalin flush. If biochemical studies need to be performed on the tissue, a 10% formalin flush should not be used as it may interfere with subsequent analysis.

2. For routine stains where perfusion is not required, tissue is sectioned and drop-fixed in a 10% formalin solution. Fixative volume should be 20 times that of tissue on a weight per volume; use 2 ml of formalin per 100 mg of tissue.

3. Due to the slow rate of diffusion of formalin (0.5 mm hr), tissue should be sectioned into 3 mm slices on cooled brain before transfer into formalin. This will ensure the best possible preservation of tissue and offers rapid uniform penetration and fixation of tissue within 3 hours.

4. Tissue should be fixed for a minimum 48 hours at room temperature.

5. After 48 hours of fixation, move tissue into 70% ethanol for long term storage.

6. Keep fixation conditions standard for a particular study in order to minimize variability. (Although set times are best, tissue may be fixed for substantially longer periods without apparent harm.

A few notes on fixation

The usual fixative for paraffin embedded tissues is neutral buffered formalin (NBF). This is equivalent to 4% paraformaldehyde in a buffered solution plus a preservative (methanol) which prevents the conversion of formaldehyde to formic acid. Because of the preservative, NBF has a shelf life of months, whereas 4% PF must be made fresh. Optimal histology requires adequate fixation, about 48 hrs at room temperature for thinly sliced tissues. Inadequately fixed tissues will become dehydrated during tissue processing, resulting in hard and brittle specimens. Alcohol based fixatives generally do not give good morphology but may be useful in special cases (such as BrdU staining). A particular challenge for the histopathology is immunostaining fixed specimens. In many cases formaldehyde fixation will prevent recognition of epitopes by the primary antibody. Occasionally, “antigen retrieval” procedures will improve results but usually frozen sections are a better bet. An alternative approach, suitable for thin or porous tissues, is to perform immunohistochemistry on fresh tissues and then post-fix and embed the tissues in paraffin.

Decalcification of bone (optional):

After fixation, bone,must be decalcified, or else it won’t cut on the microtome:

Immerse tissue cassette in 11% formic acid with a stir bar overnight in a fume hood.

Rinse in running water for 30- 60 minutes (the smell should be gone).

Storage in 70% Ethanol:
After adequate fixation tissues are transferred to 70% ethanol and may be stored at 4°C.

Paraffin infiltration

In this procedure, tissue is dehydrated through a series of graded ethanol baths to displace the water, and then infiltrated with wax. The infiltrated tissues are then embedded into wax blocks. Once the tissue is embedded, it is stable for many years.

The most commonly used waxes for infiltration are the commercial paraffin waxes. A paraffin max is usually a mixture of straight chain or n-alkanes with a carbon chain length of between 20 and 40; the wax is a solid at room temperature but melts at temperatures up to about 65°C or 70°C. Paraffin wax can be purchased with melting points at different temperatures, the most common for histological use being about 56°C–58°C, At its melting point it tends to be slightly viscous, but this decreases as the temperature is increased. The traditional advice with paraffin wax is to use this about 2°C above its melting point. To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to above 60°C or 65°C in practice to decrease viscosity.

In the schedule below, it is presumed that the working day is from 8:00 a.m. to 5:00 p.m. If other than that, appropriate adjustments should be made.

Tissue preparation
Thickness	No more than 3 mm thick.
Area	20 mm × 30 mm.
Fixed tissue	Cut large organs into 3 mm slices and store in neutral buffered formalin for 48 hours. Select tissue from fixed areas, trim to size and refix until the evening. If the trimmed sample is visibly unfixed, refix for a further 24 hours.
Unfixed tissue	Slices of tissue should be thoroughly fixed before processing.
Times	All times in processing fluids for this schedule are for tissues 3 mm thick or less. Tissues thicker than that will require longer times.
Clearing agent	Xylene or another clearing agent that will clear tissues in similar times should be used.
Processing time	This schedule takes 12 hours, and processes overnight. On weekends tissues should be left in fixative until Sunday evening with a 48 hour delay.

Trim fixed tissues and keep in neutral buffered formalin (NBF) until ready to proceed. Put tissues in a labeled (usually with pencil, as solvents dissolve the ink) cassette.

Once fixed, tissue is processed as follows, using gentle agitation, usually on a tissue processor, as follows:

1. 70% ethanol for 1 hour.

2. 95% ethanol (95% ethanol/5% methanol) for 1 hour.

3. First absolute ethanol for 1 hour .

4. Second absolute ethanol 1½ hours .

5. Third absolute ethanol 1½ hours.

6. Fourth absolute ethanol 2 hour.

7. First clearing agent ( Xylene or substitute) 1 hour.

8. Second First clearing agent (Xylene or substitute) 1 hour.

9. First wax (Paraplast X-tra) at 58°C for 1 hour.

10. Second wax (Paraplast X-tra) at 58°C 1 hour.

Due to the viscosity of molten paraffin wax, some form of gentle agitation is highly desirable. If the processor is to be run overnight it should be programmed to hold on the first ethanol bath and not finish until the next morning so the specimens do not sit in hot paraffin longer than the time indicated. If specimens are fresh they may incubate in formalin in the first stage on the machine. It is important to not keep the tissues in hot paraffin too long or else they become hard and brittle. Processed tissues can be stored in the cassettes at room temperature indefinitely.

Embedding tissues in paraffin blocks

Tissues processed into paraffin will have wax in the cassettes; in order to create smooth wax blocks, the wax first needs to be melted away placing the entire cassette in 58°C paraffin bath for 15 minutes. Turn the heat block on to melt the paraffin one hour before adding the tissue cassettes.

1. Open cassette to view tissue sample and choose a mold that best corresponds to the size of the tissue. A margin of at least 2 mm of paraffin surrounding all sides of the tissue gives best cutting support. Discard cassette lid.

2. Put small amount of molten paraffin in mold, dispensing from paraffin reservoir.

3. Using warm forceps, transfer tissue into mold, placing cut side down, as it was placed in the cassette.

4. Transfer mold to cold plate, and gently press tissue flat. Paraffin will solidify in a thin layer which holds the tissue in position.

5. When the tissue is in the desired orientation add the labeled tissue cassette on top of the mold as a backing. Press firmly.

6. Hot paraffin is added to the mold from the paraffin dispenser. Be sure there is enough paraffin to cover the face of the plastic cassette.

7. If necessary, fill cassette with paraffin while cooling, keeping the mold full until solid.

8. Paraffin should solidify in 30 minutes. When the wax is completely cooled and hardened (30 minutes) the paraffin block can be easily popped out of the mold; the wax blocks should not stick. If the wax cracks or the tissues are not aligned well, simply melt them again and start over.

The tissue and paraffin attached to the cassette has formed a block, which is ready for sectioning.Tissue blocks can be stored at room temperature for years.

Sectioning tissues

Tissues are sectioned using a microtome. Turn on the water bath and check that the temp is 35-37ºC. Use fresh deionized water (DEPC treated water must be used if in situ hybridization will be performed on the sections). Blocks to be sectioned are placed face down on an ice block or heat sink for 10 minutes. Place a fresh blade on the microtome; blades may be used to section up to 10 blocks, but replace if sectioning becomes problematic. Insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane.

Set the dial to cut 10 µM sections to order to plane the block; once it is cutting smoothly, set to 5 µM
sections . The blade should angled at 5º. Face the block by cutting it down to the desired tissue plane and discard the paraffin ribbon. If the block is ribboning well then cut another four sections and pick them up with forceps or a fine paint brush and float them on the surface of the 37ºC water bath. Float the sections onto the surface of clean glass slides. If the block is not ribboning well then place it back on the ice block to cool off firm up the wax. If the specimens fragment when placed on the water bath then it may be too hot.

Place the slides with paraffin sections on the warming block in a 65°C oven for 20 minutes (so the wax just starts to melt) to bond the tissue to the glass. Slides can be stored overnight at room temperature.

Haematoxylin Eosin (H&E) staining

Lung tissue stained with the H&E technique. Nuclei are darkly stained in this image.

H&E stain, HE stain or hematoxylin and eosin stain, is a popular staining method in histology. It is the most widely used stain in medical diagnosis; for example when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E and termed H&E section,H+E section, or HE section.

The staining method involves application ofhemalum, which is a complex formed from aluminium ions and oxidized hematoxylin. This colors nuclei of cells (and a few other objects, such as keratohyalin granules) blue. Materials colored blue by hemalum are often said to be basophilic, but this is an incorrect use of the word. The nuclear staining is folowed by counterstaining with an aqueous or alcoholic solution of eosin Y, which colors eosinophilic other structures in various shades of red, pink and orange.

Solutions:

Haematoxylin Solutions

Haematoxylin stains are commonly employed for histologic studies, often employed to color the nuclei of cells (and a few other objects, such as keratohyalin granules) blue. The mordants used to demonstrate nuclear and cytoplasmic structures are alum and iron, forming lakes or colored complexes (dye-mordant-tissue complexes), the color of which will depend on the salt used. Aluminium salt lakes are usually colored blue white while ferric salt lakes are colored blue-black.

The three main alum haematoxylin solutions employed are Ehrlich’s haematoxylin, Harris’s haematoxylin and Mayer’s haematoxylin. The name haemalum is preferable to “haematoxylin” for these solutions because haematein, a product of oxidation of haematoxylin, is the compound that combines with aluminium ions to form the active dye-metal complex. Alum haematoxylin solutions impart to the nuclei of cells a light transparent red stain which rapidly turns blue on exposure to any neutral or alkaline liquid.

Alum or potassium aluminium sulfate used as the mordant usually dissociates in an alkaline solution, combining with OH? of water to form insoluble aluminium hydroxide. In the presence of excess acid, aluminium hydroxide cannot be formed thus failure of aluminium haematoxylin dye-lake to form, due to lack of OH? ions. Hence, acid solutions of alum haematoxylin become red. During staining alum haematoxylin stained sections are usually passed on to a neutral or alkaline solution (e.g. hard tap water or 1% ammonium hydroxide) in order to neutralize the acid and form an insoluble blue aluminium haematin complex. This procedure is known as blueing.

When tap water is not sufficiently alkaline, or is even acid and is unsatisfactory for blueing haematoxylin, a tap water substitute consisting of 3.5 g NaHCO3 and 20 g MgSO4.7H2O in one liter of water with thymol (to inhibit formation of moulds), is used to accelerate blueing of thin paraffin sections. Addition of a trace of any alkali to tap or distilled water also provides an effective blueing solution; a few drops of strong ammonium hydroxide or of saturated aqueous lithium carbonate, added immediately before use, are sufficient for a 400 ml staining dish full of water. Use of very cold water slows down the blueing process, whereas warming accelerates it. In fact, the use of water below 10°C for blueing sections may even produce pink artifact discolorations in the tissue.

The staining of nuclei by hemalum does not require the presence of DNA and is probably due to binding of the dye-metal complex to arginine-rich basic nucleoproteins such as histones. The mechanism is different from that of nuclear staining by basic (cationic) dyes such as thionine or toluidine blue. Staining by basic dyes is prevented by chemical or enzymatic extraction of nucleic acids. Such extractions do not prevent staining of nuclei by hemalum.

Eosin Solutions

Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. It can be used to stain cytoplasm, collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic.Eosin is most often used as a counterstain to haematoxylin in H&E (haematoxylin and eosin) staining. Eosin stains red blood cells intensely red. Eosin is an acidic dye and shows up in the basic parts of the cell, ie the cytoplasm. For staining, eosin Y is typically used in concentrations of 1 to 5 percent weight by volume, dissolved in water or ethanol. For prevention of mold growth in aqueous solutions, thymol is sometimes added. A small concentration (0.5 percent) of acetic acid usually gives a deeper red stain to the tissue.

Other colors, e.g. yellow and brown, can be present in the sample; they are caused by intrinsic pigments, e.g. melanin.

Some structures do not stain well. Basal laminae need to be stained by PAS stain or some silver stains in order to exhibit appropriate contrast. Reticular fibers also require silver stain. Hydrophobic structures also tend to remain clear; these are usually rich in fats, eg. adipocytes, myelin around neuron axons, and Golgi apparatus membranes.

Chromosome In Situ Hybridization

2011-06-30T22:02:00.001-07:00

A modern approach to the specific location of genes on chromosomes is a technique for the hybridization of DNA and RNA "in situ." With this procedure, specific radioactive RNA or DNA (known as probes) can be isolated (or synthesized "in vitro") and then annealed to chromosomes which have been treated in such a manner that their basic double stranded DNA has been "melted" or dissociated.

In theory, and fortunately in practice, when the DNA is allowed to re-anneal, the probe competes for the binding, but only where it mirrors a complimentary sequence. Thus, RNA will attach to the location on the chromosome where the code for its production is to be found. DNA will anneal to either RNA which is still attached to a chromosome, or to the complimentary sequence DNA strand within the chromosome. Since the probe is radioactive, it can be localized via autoradiographic techniques.

Finally, it is possible to produce an RNA probe that is synthesized directly from repetitive sequences of DNA, such as that found within the nucleolar organizer region of the genome. This RNA is known as cRNA (for copied RNA) and is a convenient source of a probe for localizing the nucleolar organizer gene within the nucleus, or on a specific chromosome.

The use of in situ hybridization begins with good cytological preparations of the cells to be studied, and the preparation of pure radioactive probes for the analysis. The details depend upon whether the hybridization is between DNA (probe) and DNA (chromosome), DNA (probe) and RNA (chromosome), or between RNA (probe) and DNA (chromosome).

Preparation of the Probe:

Produce radioactive RNA by incubating the cells to be measured in the presence of H-uracil, a specific precursor to RNA. Subsequent to this incubation, extract rRNA from the sample and purify through differential centrifugation, column chromatography or electrophoresis. Dissolve the radioactive RNA probe in 4X Saline-Citrate containing 50% formamide to yield a sample that has 50,000 to 100,000 counts per minute, per 30 microliter sample, as determined with a scintillation counter. Add the formamide is added to prevent the aggregation of RNA.

Preparation of the Slides:

Fix the materials to be studied in either 95% ethanol or in 3:1 methanol:water, attach to pre-subbed slides (as squashes for chromosomes) and air dry.

Hybridization

Place the air dried slides into a moist chamber, usually a disposable petri dish containing filter paper and carefully place 30 microliters of RNA probe in 4X SSC-50% formamide onto the sample.

Carefully add a cover slip (as in the preparation of a wet mount), place the top on the container and place in an incubator at 37° C for 6-12 hours.

Washing:

Pick up the slides and dip into 2X SSC so that the coverglass falls off.

Place the slides in a coplin jar containing 2X SSC for 15 minutes at room temperature.

Transfer the slides to a treatment with RNase (50 microgram/ml RNase A, 100 units/ml RNase T1 in 2X SSC) at 37° C for 1 hour.

Wash twice in 2X SSC, 15 minutes each.

Wash twice in 70% ethanol, twice in 95% ethanol and air dry.

Autoradiography:

Add photographic emulsions to the slides and after a suitable exposure period, develop the slides, counterstain and add cover slips.

Analyze the slides by determining the location of the radioactive probe on the chromosomes or within the nuclei.

(Dr. William H. Heidcamp)

Polar Lipid Analysis

2010-12-25T06:03:00.002-08:00

From DSM Manual, courtesy Hans-Juergen Busse.

Extraction and analysis of respiratory lipoquinones.

A number of methods have been described for the extraction of respiratory lipoquinones from microorganisms. These include the use of acetone, mixtures of chlorform:methanol (2:1 v/v) or hexane:methanol (1:2 v/v). Extraction is usually carried out using freeze dried cell material, although freshly harvested or frozen cell material is used by some groups. In order to avoid loss of lipoquinone material it is avisable to carry out the extraction under nitrogen atmosphere, away from strong sunlight, and at temperatures below 35-40 C. The method described is routinely used int he author's laboratory, and permits the subsequent extraction of polar lipids from the same biomass.

1. 100 mg of freeze dried cell material is weighed into small screw cap bottles (10 ml capacity, brown glass) fitted with teflon-coated septa.

2. A small stirring bar is made from a 1 cm long piece of paper clip an also placed in the bottle.

3. To the cell material in the bottle is now added 3 ml of hexane:methanol (1:2 v/v).

4. Briefly gas the cell suspension with nitrogen and seal the bottle with the screw cap, making sure that the teflon-coated side seals against the mouth of the bottle.

5. The bottle is placed on a magnetic stirrer (about 10 such bottles can be placed on the stirrer at once) and left to stir for 30 min.

6. After 30 min stirring under nitrogen, the extracted material is placed in an ice bath until the hexane and methanol phases begin to separate.

7. Ice cold hexane (1 ml) is then added to give a methanol:hexane (1:1 v/v) biphasic mixture. Separation of the layers is enhanced by centrifuging the suspension in glass centrifuge tubes at 3000 rpm for 5 min.

8. The upper, hexane, phase is removed with a pasteur pipet and placed in a small glass tube (4-5 ml capacity).

9. The methanol phase is further extracted by the addition of cold hexane (2 ml) and 0.3% NaCl (2 ml) to give a 1:1:1 (v/v/v) ratio of hexane, methanol, and 0.3% NaCl. Centrifuge as above.

10. The upper hexane layer is then removed and added to the first hexane fraction.

11. The hexane phase is concentrated under a stream of nitrogen to give a final volume of about 0.5 ml, and the extract applied as a thin line to the lower edge of a silica gel plate (containing a fluorescent indicator F254).

12. The plates are developed in hexane:tert-butylmethylether (9:1 v/v) until the solvent front reaches the upper edge of the plate (20-30 min).

13. The developed plate is examined under UV light (254 nm) briefly to locate respiratory lipoquinones, which appear as dark bands against a greenish background. Menaquinones have an Rf value of about 0.7, while ubiquinones have an Rf of about 0.4.

14. The lipoquinone material is removed from the silica gel by scraping the appropriate area from the thin layer plate and placing it in a narrow bore tube (or pasteur pipet) fitted with a frit or glass wool plug. After tapping the tube to settle the silica gel, the lipoquinone material is eluted by passing 2x 0.5 ml of hexane:methanol (1:2 v/v) through the column, the eluant being collected in a small glass bottle (1.5 ml capacity). The purified lipoquinones may be recovered into hexane by cooling the mixture until phase separation occurs, followed by the addition of a few drops of 0.3% NaCl and about 0.3 ml of cold hexane. The upper, hexane phase containing the lipoquinones, is transferred to a second small 1.5 ml glass bottle, and may be stored at -20 C for analysis by UV spectroscopy, HPLC, or mass spectroscopy.

15. Further analysis of the lipoquinones is carried out by evaporating the hexane phase. HPLC analysis of the lipoquinones is carried out by dissolving the material in 100-200 ul of the HPLC solvent. UV spectroscopy is carried out by dissolving eh lipoquinone in a suitable volume (usually 0.5 - 1 ml) of spectroscopic grade hexane, ethanol, or (more commonly) iso-octane. Mass spectra of lipoquinones are recorded on material dissolved in the minimum quantity of a suitable solvent, such as methanol.

UV Spectroscopy

UV Spectra of respiratory lipoquinones give information on the nature of the quinone nucleus. UV spectra may be recorded in hexane, ethanol or iso-octane (the latter being the most frequently used). Spectra are recorded between 200 nm and 500 nm in quartz cuvettes. Menaquinone type lipoquinones (naphthoquinones) give four maxima between 230 nm and 280 nm, while ubiquinone type lipoquinones (benzoquionones) give two maxima lying very close together between 260 nm and 280 nm.

Polar Lipid Extraction

Polar lipids are generally extracted from dry cell material using chloroform:methanol:0.3% NaCl (1:2:0.8 v/v/v). This may be carried out by adding 9.5 ml of this mixture to 100 mg of freeze dried cells, or by adding a suitable amount of chloroform, methanol and 0.3% NaCl to the cell material, or to the aqueous methanolic phase remaining from the lipoquinone extraction.

1. The aqueous methanolic phase (4 ml total volume), together with the cell material from the lipoquinone analysis, is diluted with 5.5 ml of Chloroform:Methanol (2.5:3.0 v/v) to give a chloroform, methanol, 0.3% NaCl (1:2:0.8 v/v/v) mixture.

2. The mixture is placed in a 15 ml bottle with a teflon lined screw cap (check that the magnetic stirrer is in the bottle), gassed briefly with nitrogen, sealed and heated for 15 min at 80 C (with occasional shaking).

3. Allow the mixture to cool to room temperature on a magnetic stirrer. Check that the mixture is homogeneous, the presence of excess hexane will cause phase separation and may be overcome by adding a small amount of methanol until a homogeneous mixture is obtained.

4. The cell debris is removed by centrifugation in glass centrifuge tubes, at 3000 rpm for 5-10 min, and the supernatant decanted into 5 ml of chloroform:0.3% NaCl (1:1 v/v). The latter mixture (2.5 ml chloroform and 2.5 ml of 0.3% NaCl) may be placed in the extraction buffer bottles after they have been washed with distilled water.

5. After brief mixing, the biphasic mixture is centrifuged in glass centrifuge tubes (3000 rpm for 5 min) and the lower, chloroform phase collected with a pasteur pipet. Take care not to remove the flocculent protein layer or the aqueous phase.

6. The chloroform phase, containing the polar lipids is taken to dryness under a stream of nitrogen (when using a heating block or a water bath to accelerate drying, do not exceed 40 C).

7. The dried lipid material is then re-dissolved in 250 ul of chloroform:methanol (2:1 v/v) and transferred toa small glass bottle or ampule and may be stored for periods of several months at temperatures of -20 C or lower.

8. The lipid solution is used to spot thin layer plates; the lipids being placed in the bottom left-hand corner of each plate (Fig 4-A). Glass or aluminum plates are used because many detection reactions require the plates to be heated.

Run the first dimension with the spotted lipid on the lower right hand corner. After drying, run the plates in the second dimension with the lipid in the lower left hand corner.

9. Develop the plates in two dimensions using, in the first dimension chloroform:methanol:water (65:25:4 v/v/v), and in the second dimension, chloroform:methanol:acetic acid:water (80:12:15:4 v/v/v/v). Between the two dimensions, the plates should be dried at room temperature for about 20-30 min.

10. Lipid functional groups are identified using spray reagents specific for phosphate (Zinzadze), alpha-glycols (periodate-Schiff), and sugars (alpha-naphthol/H2SO4, anisaldehyde/H2SO4), free amino groups (ninhydrin), quaternary nitrogen (Dragendorff), and primary and secondary amines (chlorine and starch/iodide). Identification of the various lipids is carried out on the basis of staining reaction and Rf values. All spraying must be carried out in a fume hood using sufficient ventilation, since all lipid spray reagents are toxic.

Retrieved from "http://www.openwetware.org/wiki/Polar_Lipid_Analysis"

Mitochondrial DNA Isolation

2010-12-25T06:03:00.001-08:00

What's mitochondrial DNA? mitochondrial DNA (mtDNA) is a genetic material of the mitochondria, the organelles that generate energy for the cell. In mammals, mtDNA makes up less than 1% of the total cellular DNA, but in plants the amount is variable. It codes for ribosomal RNA and transfer RNA, but only some mitochondrial proteins (up to 30 proteins in animals), the nuclear DNA being required for encoding most of these. Mitochondrial DNA which is passed down from the mother to all her children, males and females

Protocol 1. Mitochondrial DNA Isolation (BioVision)

Protocol 2. Mitochondrial DNA Isolation

Protocol 3. Comparison of different methods for extraction of mitochondrial ...

quality of DNA. Some reports have described obtaining mtDNA. using techniques based on the preparation of spheroplastis or. isolation of mitochondria (6,15 ...
www.nih.go.jp/JJID/55/122.pdf

Protocol 4. Purification of mitochondrial DNA.

A minimum of 2-3 g wet weight of cells are necessary for one DNA extraction (preferably 10-20 g). The cells are harvested in the early stationary phase by ...
www.bch.umontreal.ca/People/lang/FMGP/methods/mtDNA.html

Protocol 5. Fossil Hominids: mitochondrial DNA

1997) claimed to have extracted mitochondrial DNA (mtDNA) from a piece of bone cut from the upper arm of the first recognised Neandertal fossil, ...
www.talkorigins.org/faqs/homs/mtDNA.html

Protocol 6. Mitochondrial DNA Analysis at the FBI Laboratory (Topical List ...

Backgroung of mitochondril DNA

Current problems

Test procedures

Database

Other fosenic information related to mitochondrial DNA

Microsatellite Protocols

2010-12-25T06:02:00.000-08:00

What’s microsatellites ?

A DNA sequence from 2 to 6 nucleotides which are tandemly repeated from about 5 to 5,000 times (usual range 20 - 50 repeats). Microsatellites are found at many different loci in the genome, including both coding and non-coding regions. A locus containing a microsatellite is often polymorphic because of variation in the repeat number.

Protocols

Optimization of PCR Protocol in Microsatellite Analysis with ...
Here we present the optimization of PCR conditions for microsatellite analysis. of coniferous trees. The use of touchdown protocol for annealing resulted in ...
pubs.nrc-cnrc.gc.ca/ispmb/ispmb18/R00-053.pdf

Microsatellite Cloning Protocol (June 2001)

DeWoody‘s Microsatellite Cloning Protocol (Spring 2002) ..... Microsatellite clones in the kangaroo rat. Using the protocol described above, ...
www.agriculture.purdue.edu/fnr/html/faculty/DeWoody/DeWoodyweb/pdfs/msatclngprtcl.pdf

How Are Microsatellite Repeats Counted?

How Microsatellite Repeats Are Counted ] [ YSTR Database Allele Frequency ... at http://www.ihwg.org/protocols/hct/HCT-microsatellite-protocol.pdf which may ...
www.contexo.info/DNA_Basics/microsatellite_analysis.htm

MHC MICROSATELLITE TYPING PROTOCOL

File Format: Microsoft Word - View as HTML
MHC MICROSATELLITE TYPING PROTOCOL. SPECIMEN:. Genomic DNA from any source such as whole blood, peripheral blood lymphocytes, serum, or plasma can be used. ...
www.ihwg.org/protocols/hct/MS.doc

Microsatellites Protocol

In order to identify the microsatellites on the Pacu fish, the following protocol had to be followed. Step 1: Extracting the DNA from the nucleated blood ...
www.woodrow.org/teachers/esi/2002/Biology/Projects/p3/protocol.htm

Microsatellite library protocol

The following protocol uses the microsatellite hybrid capture technique for isolating microsatellite loci [Prochazka 1996, Brown et al., 1995]. ...
hcgs.unh.edu/protocol/msat/

Microsatellite Library Protocol Last Update 11/26/03 D Drown ...
Microsatellite Library Protocol. Last Update 11/26/03 D Drown. Modified from T. Garner Protocol. Outline of Protocol. DNA Extraction (1 overnight [optional] ...
people.bu.edu/pbarber/Web%20Protocols/Microsatellite%20Library.pdf

SOLTIS LAB MICROSATELLITE CAPTURE PROTOCOL

This first step in the microsatellite capture protocol uses restriction enzymes to cut. genomic DNA into smaller fragments. We will be using Sau3A I, ...
www.flmnh.ufl.edu/soltislab/Soltis_SSR_Capture.pdf

Science Links Japan | A Protocol for Efficient Development of MicrosatelliteMarkers.

Title;A Protocol for Efficient Development of Microsatellite Markers. Author;LIAN C(Univ. Tokyo, Asian Natural Environmental Sic. ...
sciencelinks.jp/j-east/article/200414/000020041404A0421669.php

Microsatellite Protocol

Microsatellite Protocol. Preparation of reaction mixture. Dilute primers to 10 µM by adding 200 µl 1x TE buffer (see Table 8-9). Each primer in the Rice Map ...
www.pustaka-deptan.go.id/rkb/knowledgeBank/grcOpsManual/_Microsatellite_Protocol.htm - 50k - Cached - Similar pages

Isolating Microsatelline DNA Loci*

File Format: PDF/Adobe Acrobat - View as HTML
In theory, this protocol will. work for any eukaryotic organism (i.e, anything with an appreciable number of microsatellite ...
www.uga.edu/srel/DNA_Lab/Glenn&Schable_update2.pdf

Microsatellite Protocol.
Microsatellite Protocol. Prepare gel:. 1. Pour Acrylamide gel (mix up 25 mL Acrylamide solution, 125 uL APS, 17 uL. TEMED) ...
people.bu.edu/pbarber/Web%20Protocols/Microsatellite%20gel%20protocol.pdf -

Fully automated DNA purification and efficient multiplex PCR for ...
concentration do not significantly influence. multiplex PCR of microsatellite loci. Table 1. PCR cycling protocol for microsatellite analysis ...
www1.qiagen.com/literature/qiagennews/0103/1023229_QNews12003_p08_10.pdf

Microsatellite Protocol - 30nov
flanking primers for microsatellite amplification. This protocol greatly improves. enrichment by increasing the efficiency of linker ligation to genomic DNA ...
www9.georgetown.edu/faculty/hamiltm1/Downloads_files/SNXmicro11.pdf

SilkSatDB-A Silkworm Microsatellite Database

Protocol for analysing microsatellites using agarose gel electrophoresis, is a fast and efficient technique for standardising primers and quick polymorphism ...
210.212.212.7:9999/PHP/SILKSAT/index.php?f=protocol_ssr

Rob Toonen's Protocol （for Microsatellite）

Estoup & Turgeon's Microsatellite Protocols （for Microsatellite）

Fisher & Bachmann's Protocol （for Microsatellite）

Theresa M. Culley (DNA Based Methods / Microsatellite)

Dec 30, 2005 ... If you are interested in doing this process yourself, you will need to email Travis for the most current microsatellite development protocol ...
www.biology.uc.edu/faculty/culley/Protocols.htm

NOTES Microsatellite Multiplexing in Fish
Step-by-step protocol for the design and optimization of microsatellite multiplexing (modified from ..... mized microsatellite multiplex protocol presented ...
publish.uwo.ca/~bneff/papers/microsatellite_multiplexing_in_fish.pdf

Insect Microsatellite Database

LINKS. Silkworm microsatellite database · Silkbase · Silkworm science/Sericulum · Bombyx mori genome map information system · Silkworm, Nucleic acid/Amino ...
210.212.212.8/PHP/INSATDB/links.php

Hop DNA Extraction Protocol

2010-12-25T06:01:00.000-08:00

1. Obtain an adequate amount (~ 1g) of fresh hop leaves and crush them with liquid Nitrogen and a small amount of Carborundum powder (fine 320 grit).

2. Assume 90% of mass is water weight.

3. Add 3.3 ml of buffer per gram of wet (16 ml per gram of dried) hop leaves, and incubate for 1 to 4 hours at 60-65°C.

4. Transfer 900 μl into fresh tube

5. add 600 μl of 24:1 CHCl3:octanol and invert gently (do NOT vortex!).

6. Centrifuge at 5000g for 10 minutes.

7. Transfer supernatant (800 μl) into new 2-ml tube.

8. Add 5μl of RNAase and incubate at 37°C for 30 minutes (or more).

9. Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.

10. Spin down for 10 min. at RT.

11. Add 500 μl wash buffer and incubate 10 min. at RT.

12. Carefully remove wash buffer. Don't lose DNA pellet!

13. Briefly centrifuge to collect pellet at bottom of tube - remove any remaining wash buffer.

14. Dry pellet at RT or 50°C to speed up.

15. Add 100 μl ddH2O to dissolve DNA.

16. Store at -20°C until needed.

17. Run electrophoresis for analysis.

Prepared solutions

1. Buffer: 100 ml: 50 mM Tris/HCl (ph 8.0), 1.8 M NaCl, 50 mM EDTA. Then add 10 mg/ml of CTAB ( 200 mg per 20 ml buffer, final conc. = 1%) and 1 μl/ml 2-mercaptoethanol (20 μl to 20 ml buffer; final conc. = 0.1%).

2. Wash buffer 100 ml: 200 μl 5M NH4OAc (final conc. = 10 mM), 76.0 ml abs. ethanol (final conc. = 76%), and 23.8 ml of sterilized water.

Cell Cycle Assay by PI

2010-12-25T05:59:00.001-08:00

Cancer Research UK - PI Staining Protocol

Propidium Iodide Staining. Harvest cells in the appropriate manner and wash in PBS. ... This protocol is also available in PDF format. ...
science.cancerresearchuk.org/.../cell_cycle_analysis/

BrDU-PI Staining

0.08% w/v Pepsin: Dissolve 0.4 g pepsin in 500 mL of 0.1M HCl (496 ml dd H20 + 4.1 mL conc. HCl), filter, store 4ºC. ...
www.fhcrc.org/science/labs/fero/Protocols/BrDU_PI.html

Cell Cycle Analysis by Propidium Iodide

Cell Cycle Analysis by Propidium Iodide (PI) Staining. Adherent cells:. trypsinized; suspended in medium + 10% FCS; centrifuged (1000 rpm, 5 min) ...
www.meduniwien.ac.at/user/johannes.schmid/PIstaining3.htm

Ethanol-fixation of Samples for Long-term Storage and Subsequent DNA Staining Ethanol

Ethanol-fixation of Samples for Long-term Storage and Subsequent DNA Staining. I. Materials. 70% Ethanol at – 20oC. DNA Staining Buffer: ...
cyto.mednet.ucla.edu/Protocols/ethanol.htm

FIXATION and DNA Staining for Cell Cycle Analysis

Flow Cytometry Facility at Iowa State University

FIXATION and DNA Staining for Cell Cycle Analysis. Background. This method of DNA staining utilizes ethanol to fix the cells and permeabilize the membrane, ...
www.biotech.iastate.edu/facilities/CELLHYB/CCAnalysis.htm

Preparing Cells for FACS/PI Analysis (Cell Cycle Analysis)

www.flemingtonlab.com. Preparing cells for PI/FACS (cell cycle) analysis. Experimental design considerations. - This method works well to assess cell cycle ...
www.flemingtonlab.com/Protocols/PreparingCellsforFACS-PI.pdf

The SickKids-UHN Flow Cytometry Facility - DNA Staining for Flow ...

DNA/PI Staining for Cell Cycle · Extracellular Staining Protocol ... Propidium iodide solution: 0.1mg/ml PI (Sigma) in HBSS with 0.6% NP-40. HBSS Staining ...
www.sickkids.ca/fcf/custom/DNAstain.asp

PI Staining of Dead Cells

PROPIDIUM IODIDE STAINING OF DEAD CELLS FOR FLOW CYTOMETRY. Propidium iodide (PI) intercalates into double-stranded nucleic acids. ...
cyto.mednet.ucla.edu/Protocols/pi.htm

PI Staining - - University of Arkansas for Medical Sciences

UAMS, Immunoflourescence Staining for Immunotyping · Propdium Iodide (PI) Staining · Sorting and Staining Buffers · 2% Paraformaldehyde ...
www.uams.edu/flowcytometry/folderone/PI.asp

Cell Cycle

While PI staining provides a very precise image of the cellular state with ... For your convenience, we have placed a BRDU staining protocol on the core web ...
www.med.umich.edu/flowcytometry/InitialTraining/lessons/lesson6/index.htm

Flow Cytometric Protocols for Cell Cycle Analysis

Protocol for staining whole cells with PI:. 1. Harvest cells and prepare single cell suspension in buffer (e.g. PBS + 2% FBS; PBS + 0.1% BSA) ...
www.molbio1.princeton.edu/facility/flowcyt/cycle1.html

Von Kossa Stain Protocol

2010-11-09T05:25:00.000-08:00

The method was originally developed by von Kossa, and underwent several modifications. The stain principle is a precipitation reaction in which silver ions react with phosphate (not calcium) in the presence of acidic material. Photochemical degradation of silver phosphate to silver then occurs under light illumination.

Additional methods need to be employed to confirm the presence of calcium and hydroxyapatite, such as Alizarin Red, which detects calcium deposits.

VON KOSSA Stain Procedure:

1. Sections to water.

2. Place sections in 1% silver nitrate solution under reasonable light for 30-60 minutes.

3. Wash in distilled water.

4. Treat with 3% sodium thiosulphate for 3-5 minutes.

5. Wash in water.

6. Counterstain with van Gieson for 3-5 minutes.

7. Wash in alcohol.

8. Clear .

9. Mount sections

RESULTS:

Mineralised bone black Osteoid red

Other Protols for Von Kossa

von Kossa Staining Protocol for Calcium

von Kossa Staining Protocol for Calcium. NovaUltra Special Stain Kits. Description: This technique is for demonstrating deposits of calcium or calcium salt ...
www.ihcworld.com/_protocols/special_stains/von_kossa.htm

Von Kossa Stain - Wikipedia, the free encyclopedia

Von Kossa Stain. From Wikipedia, the free encyclopedia. Jump to: navigation, search. Thevon Kossa stain is used to quantify mineralization in cell culture ...
en.wikipedia.org/wiki/Von_Kossa_Stain

Von Kossa's Method - Calcium
VON KOSSA'S METHOD - CALCIUM. PURPOSE: Abnormal deposits of calcium may be found in any area of the body. With the H&E stain, calcium appear deep ...
library.med.utah.edu/WebPath/.../VONKOSSA.PDF

Renal Fellow Network: the von Kossa Stain for Acute Phosphate ...

17 Aug 2009 ... A good detection method is the use of the von Kossa stain, which stains certain calcium-containing salts such as calcium phosphate a ...
renalfellow.blogspot.com/.../von-kossa-stain-for-acute-phosphate.html

Technical Bulletin #444 von Kossa Staining of Osteoclast ...
von Kossa Staining of Osteoclast Resorption on BD BioCoat™ Osteologic™ Discs ... Figure 1: von Kossa stain of BD BioCoat™ Osteologic™ Disc following ten-day ...
www.bdbiosciences.com/external_files/dl/doc/tech.../TB444.pdf

von Kossa staining alone is not sufficient to confirm that ...
Numerous techniques are currently used to characterize biological mineralization in intact tissues and cell cultures; the von Kossa staining method, ...
www.ncbi.nlm.nih.gov/pubmed/12724828

The von Kossa reaction for calcium deposits: silver lactate ...
We have demonstrated that positive von Kossa stain- ing correlates well with calcifications in... cultures the von Kossa stain usually yielded identifiable ...
www.springerlink.com/index/k782p815tn252t39.pdf

Re: [Histonet] von kossa stain

27 Jan 2004 ... My understanding of the von Kossa stain is that it is NOT specific for calcium. Rather, the silver nitrate in the staining solution (silver ...
www.histosearch.com/histonet/.../Re.Histonetvonkossastain.html

VON KOSSA STAIN KIT PROCEDURE
VON KOSSA STAIN KIT PROCEDURE. Item# KT 028. KIT. 100ml. Nuclear Fast Red Stain. 100ml. 5% Silver Nitrate. COMPONENTS. 100ml. 5% Sodium Thiosulfate ...
search.cosmobio.co.jp/cosmo_search_p/..._/KT028.20060119.pdf

von Kossa stain - Definition

von Kossa stain definition from the mondofacto online medical dictionary.
www.mondofacto.com/facts/dictionary?von+Kossa+stain

Von Kossa Staining Alone Is Not Sufficient to Confirm that ...
and cell cultures; the von Kossa staining method, elec- tron microscopic analysis (EM), ....However, the von Kossa stain is being used with greater ...
www.springerlink.com/index/TWUFXJY1VTHUVYR5.pdf

von Kossa and Alcian Blue Staining Protocol for Calcium and ...

Combination of von Kossa and Alcian Blue staining will make both calcium and mucosubstances visible on the same tissue sections. ...
www.ihcworld.com/_...stains/von_kossa.alcian_blue.htm

Histology Lab: VON KOSSA

Histotechnology Technical methods. Stain for calcium ... VON KOSSA. 1. Sections to water. 2. Place sections in 1% silver nitrate solution under ultra- ...
www.nottingham.ac.uk/pathology/protocols/vonkoss.html

Von kossa stain: Definition with Von kossa stain Pictures and Photos

Definition of Von kossa stain with photos and pictures, translations, sample usage, and additional links for more information.
www.lexic.us/definition-of/von_kossa_stain

Stain Expert™ - Your Stains & Reagents Information Center

Calcium Stain - Von Kossa. Celestine Blue Method - Lendrum. Copper Stain (Rubeanic Acid). Cresyl Violet Acetate Method for Helicobacter sp. ...
www.sigmaaldrich.com

JoVE: Undecalcified Bone Preparation for Histology ...
8 Jan 2010 ... An identical protocol or rack staining is necessary for ... Von Kossa. Place in silver nitrate solution and expose to strong light until ...
www.jove.com/index/details.stp?id=1707

Isolation of human multipotent mesenchymal stem cells from second ...
We developed a novel two‐stage culture protocol for isolating MSCs from amniotic .... (d) Osteogenic differentiation was demonstrated by von Kossa staining. ...
humrep.oxfordjournals.org/content/19/6/1450.full

BioMed Central | Full text | Isolation of mesenchymal stem cells ...
30 May 2007 ... Von Kossa staining of calcium-phosphate deposits was performed. ... (1999) [40] and a slightly modified protocol of the one reported by ...
www.biomedcentral.com/1472-6750/7/26

Fwd: [Histonet] Question on Von Kossa

Of course, as with any silver stain use acid cleaned glassware and gloves ... Does anyone have a good Von Kossa stain protocol that they would not mind ...
www.mail-archive.com/histonet@lists...edu/msg00672.html

Telomere and telomerase (2)

2010-01-15T21:38:00.001-08:00

Introduction to Telomeres and Telomerase

Author(s): Michael C. Bibby

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:01; Page Range: 1-12

Abstract | Full Text | PDF (81K)

Detection of Chromosome Ends by Telomere FISH

Author(s): Harry Scherthan

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:13; Page Range: 13-31

Abstract | Full Text | PDF (117K)

Telomere Length Distribution: Digital Image Processing and Statistical Analysis

Author(s): Jean-Patrick Pommier, Laure Sabatier

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:33; Page Range: 33-63

Abstract | Full Text | PDF (351K)

Analysis of Telomerase RNA Gene Expression by In Situ Hybridization

Author(s): W. Nicol Keith, Joseph Sarvesvaran, Martin Downey

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:65; Page Range: 65-81

Abstract | Full Text | PDF (132K)

Relative Gene Expression in Normal and Tumor Tissue by Quantitative RT-PCR

Author(s): Dennis S. Salonga, Kathleen D. Danenberg, Jean Grem, Ji Min Park, Peter V. Danenberg

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:83; Page Range: 83-98

Abstract | Full Text | PDF (138K)

Quantitative Detection of Telomerase Components by Real-Time, Online RT-PCR Analysis with the LightCycler

Author(s): Thomas Emrich, Sheng-Yung Chang, Gerlinde Karl, Birgit Panzinger, Chris Santini

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:99; Page Range: 99-108

Abstract | Full Text | PDF (98K)

Standard TRAP Assay

Author(s): Angelika M. Burger

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:109; Page Range: 109-124

Abstract | Full Text | PDF (131K)

Stretch PCR Assay

Author(s): Jun-ichi Nakayama, Fuyuki Ishikawa

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:125; Page Range: 125-136

Abstract | Full Text | PDF (110K)

Fluorescent Detection of Telomerase Activity

Author(s): Wade K. Aldous, Amber J. Marean, Mary J. DeHart, Katherine H. Moore

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:137; Page Range: 137-146

Abstract | Full Text | PDF (99K)

Nonradioactive Detection of Telomerase Activity Using a PCR-ELISA-Based Telomeric Repeat Amplification Protocol

Author(s): Thomas Emrich, Gerlinde Karl

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:147; Page Range: 147-158

Abstract | Full Text | PDF (123K)

In Situ TRAP Assay Detection of Telomerase Activity in Cytological Preparations

Author(s): Kazuma Ohyashiki, Junko H. Ohyashiki

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:159; Page Range: 159-164

Abstract | Full Text | PDF (143K)

Biotinylated Primer for Detecting Telomerase Activity Without Amplification

Author(s): Daekyu Sun

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:165; Page Range: 165-171

Abstract | Full Text | PDF (81K)

Whole-Cell and Microcell Fusion for the Identification of Natural Regulators of Telomerase

Author(s): Henriette Gourdeau, Marsha D. Speevak, Lucie Jetté, Mario Chevrette

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:173; Page Range: 173-195

Abstract | Full Text | PDF (145K)

Screening with COMPARE Analysis forTelomerase Inhibitors

Author(s): Imad Naasani, Takao Yamori, Takashi Tsuruo

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:197; Page Range: 197-207

Abstract | Full Text | PDF (125K)

Telomerase as a Therapeutic Target: Therapeutic Potential of Telomerase Inhibitors

Author(s): John A. Double

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:209; Page Range: 209-216

Abstract | Full Text | PDF (71K)

Methods of Telomerase Inhibition
Additionally, methods are outlined in this chapter for determining the effectiveness oftelomerase inhibition through TRAP assays or assessment of telomere ...
www.ncbi.nlm.nih.gov/pmc/articles/PMC2423206/

Telomere and telomerase (1)

2010-01-15T21:37:00.000-08:00

Telomere and telomerase assay

A telomere is a region of repetitive DNA at the end of a chromosome, which protects the end of the chromosome from deterioration. Its name is derived from the Greek nouns telos (τἐλος) "end" and merοs (μέρος, root: μερ-) "part".

Russian theorist Alexei Olovnikov was the first to recognize (1971) the problem of how chromosomes could replicate right to the tip, as such was impossible with replication in a 5' to 3' direction. To solve this and to accommodate Leonard Hayflick's idea of limited somatic cell division, Olovnikov suggested that DNA sequences would be lost in every replicative phase until they reached a critical level, at which point cell division would stop.

During cell division, the enzymes that duplicate the chromosome and its DNA cannot continue their duplication all the way to the end of the chromosome. If cells divided without telomeres, they would lose the ends of their chromosomes, and the necessary information they contain. (In 1972, James Watson named this phenomenon the "end replication problem".) The telomeres are disposable buffers blocking the ends of the chromosomes and are consumed during cell division and replenished by an enzyme, the telomerase reverse transcriptase.

Elizabeth Blackburn compared telomeres to the aglets (tips) on the ends of shoelaces that keep them from fraying.

In 1975–1977, Blackburn, working as a postdoctoral fellow at Yale University with Joseph Gall, discovered the unusual nature of telomeres, with their simple repeated DNA sequences composing chromosome ends. Their work was published in 1978. The telomere shortening mechanism normally limits cells to a fixed number of divisions, and animal studies suggest that this is responsible for aging on the cellular level and sets a limit on lifespans. Telomeres protect a cell's chromosomes from fusing with each other or rearranging—abnormalities which can lead to cancer—and so cells are normally destroyed when their telomeres are consumed. Most cancers are the result of "immortal" cells which have ways of evading this programmed destruction.

Elizabeth Blackburn, Carol Greider, and Jack Szostak were awarded the 2009 Nobel Prize in Physiology or Medicine for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.

Telomeres and telomerase: biological and clinical importance ...
(15) developed an assay for testing telomerase activity in cell extracts. Based on identification of telomerase mechanisms and properties, the telomere ...
www.clinchem.org/cgi/content/full/43/5/708

Telomere Length Analysis and In Vitro Telomerase Assay.

1 Introduction; 2 Materials and Methods; 2.1 Telomere Measurement; 2.2 Telomere Cloning; 2.3 TRAP Telomerase Assay; 3 Notes; References. Browse by Subject ...
www.springerprotocols.com/Abstract/doi/10.../1-59259-434-4:123

Facts about Telomeres and Telomerase

The following section will teach you the basics of telomeres and telomerase. It will also introduce you to the potential applications of current telomerase ...
www4.utsouthwestern.edu/cellbio/shay.../sw_facts.html

TRAP–LIG, a modified telomere repeat amplification protocol assay to quantitate telomerase inhibition by small molecules.
Its major function is to maintain the length of telomeric DNA by synthesizing telomeric DNA repeats, and its enzymatic activity is assayed by means of the ...
linkinghub.elsevier.com/retrieve/pii/S0003269708002960

Telomeres and Telomerase: The Cellular Timekeepers

The telomeres appear to be the cellular timekeepers, but are they related to human aging?
www.senescence.info/telomeres.html

Effect of Telomere and Telomerase Interactive Agents on Human Tumor and Normal Cell Lines.
The cytotoxic effect of telomere and telomerase interactive agents against normal human marrow cells in the colony-forming assay showed the same range of ...
clincancerres.aacrjournals.org/content/6/3/987.full

Cell Research - Telomere and telomerase in oncology
Shortening of the telomeric DNA at the chromosome ends is presumed to limit the lifespan of... Telomere, telomerase, cancer, telomerase assay, inhibitor ...
www.nature.com

Telomeres and telomerase — Genes & Development
Cdc13p (gray) and Est1 (purple) may recruit telomerase to the telomeric 3′ end in ..... (1995) An in vitro assay for Saccharomyces telomerase requires EST1. ...
genesdev.cshlp.org/content/13/18/2353.full

Telomerase Assay in Renal Cancer

Bacchetti, S. and Counter, C. M. (1995) Telomeres and telomerase in human cancer. ...quantitative nonisotopic assay for telomerase activity in humantumors. ...
www.springerprotocols.com/Abstract/doi/10.1385/1-59259-144-2:05

Protein Purification Related Articles

2010-01-15T21:36:00.000-08:00

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1. Expression and Purification of Soluble and Correctly Folded Bovine β-Lactoglobulin Variants A and B in Escherichia coli for NMR Studies
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 16 December 2009
Komala Ponniah, Trevor S. Loo, Patrick J.B. Edwards, Steven M. Pascal, Geoffrey B. Jameson, ...

2. Expression of soluble and functional human neonatal Fc receptor in Pichia pastoris
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 16 December 2009
Chang-Han Lee, Dong-Ki Choi, Hye-Ji Choi, Moo-Young Song, Yong-Sung Kim

3. Formate-nitrate transporters: optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 16 December 2009
Katherine S.H. Beckham, Jane A. Potter, Shiela E. Unkles

4. Streamlined, automated protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) using ÄKTAxpress™
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 6 December 2009
Cornelia Ludwig, Martin A. Wear, Malcolm D. Walkinshaw

5. Expression, purification and primary crystallographic study of human androgen receptor in complex with DNA and coactivator motifs
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 6 December 2009
X. Edward Zhou, Kelly Suino-Powell, Phumzile L. Ludidi, Donald P. McDonnell, H. Eric Xu

6. Purification and characterization of recombinant CH3 domain fragment of the CREB-binding protein
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 6 December 2009
Catherine Ibarra Drendall, Quang H. Pham, Eric C. Dietze

7. Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Trang Thi Phuong Phan, Hoang Duc Nguyen, Wolfgang Schumann

8. An Improved Procedure for the Purification of the Escherichia coli RNA Polymerase Omega Subunit
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Catherine E. Vrentas, Tamas Gaal, Richard R. Burgess, Richard L. Gourse

9. Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Trang Thi Phuong Phan, Hoang Duc Nguyen, Wolfgang Schumann

10. An Improved Procedure for the Purification of the Escherichia coli RNA Polymerase Omega Subunit
Publication year: 2009
Source: Protein Expression and Purification, In Press, Accepted Manuscript, Available online 4 December 2009
Catherine E. Vrentas, Tamas Gaal, Richard R. Burgess, Richard L. Gourse

Protein Purification (from Wikipedia, the Free Encyclopedia)

Contents

1 Purpose

2 Strategies

3 Evaluating purification yield

4 Purification of a tagged protein

5 Methods of protein purification

6 Extraction

7 Precipitation and differential solubilization

8 Ultracentrifugation

9 Chromatographic methods

9.1 Size exclusion chromatography

9.2 Separation based on charge or hydrophobicity

9.3 Ion exchange chromatography

9.4 Affinity chromatography

9.4.1 Metal binding

9.4.2 Immunoaffinity chromatography

9.5 HPLC

10 Concentration of the purified protein

10.1 Lyophilization

10.2 Ultrafiltration

11 Analytical

11.1 Denaturing-Condition Electrophoresis

11.2 Non-Denaturing-Condition Electrophoresis

12 References

13 External links

Protein Purification and Identification Protocols

2010-01-15T21:34:00.000-08:00

Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterisation of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity.

1. ALKYL Aspartamide HIC Columns

2. Comparison of affinity tags for protein purification.

3. DEAE Column Preparation

4. DEAE column: FPLC

5. Green Fluorescent Protein Isolation (Gary J. Lindquester)

6. GST Fusion Protein Preparation

7. GST Fusion Protein Purification from Yeast

8. GST-Fusion Protein Purification

9. HeLa Cell Nuclei Preparation (John Garland, The Biochemistry and Molecular Biology Department of The James H. Quillen College of Medicine in Johnson City, TN)

10. HILIC and SEC Columns PolySULFOETHYL and PolyHYDROXYETHYL Columns

11. Isolation of Protein from Tissue (Reddy research laboratory, Neurological Sciences Institute, Oregon Health & Science University)

12. Making Affinity Columns

13. Making Conductivity Curves (Waters Lab)

14. PI-PLC Purification (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

15. Polyaspartic Acid Weak Cation Exchange Columns

16. PolyGLYCOPLEX Complex Carbohydrates Columns

17. PolySULFOETHYL Aspartamide Strong Cation Exchange Columns

18. PolyWAX LP Weak Anion Exchange Columns

19. Preparation of Tonsil Lysate (Springer Lab, Harvard University)

20. Protein Extraction (Frank Bottone Jr.)

21. Protein Extraction from Tissues (Reddy research laboratory, Neurological Sciences Institute, Oregon Health & Science University)
Protein Isolation Protocols (Lamond Lab, University of Dundee)

22. Protein Isolation using Trizol (Frank Bottone Jr.)

23. Protein Purification

24. Protein Purification

25. Protein Purification

26. Protein Purification

27. Protein Purification (handbook)

28. Protein Purification (Michael Blaber)

29. Protein Purification (Michael Blaber)

30. Protein Purification (Michael Blaber)

31. protein purification (Pictures)

32. Protein Purification and Analysis Protocols and Applications Guide (Promega)

33. Protein Purification on 1G3 Column (Bjorkman Group, Howard Hughes Medical

34. Protein Purification Protocols (The Protein Expression and Purification Facility, EMBL)

35. Protein Sample Preparation & Protein Purification (Invitrogen)

36. Purificaiton of Protein by Affinity Chromatography (Krause Lab)

37. Purification of GST Fused Proteins

38. Purification of GST Fused Proteins

39. Purification of His-tagged proteins from Sf9 cells under native conditions

40. Purification of Proteins (NCBI)

41. Purification Scheme for Ubiquitin Expression Lysate (Sosnick Lab, University of Chicago)

42. Purification of Maltose Binding Proteins Fused Proteins

43. Purification of MBP (maLTose-Binding Proteins) Fused Proteins (Bowtell Lab

44. Removal of Nucleic Acids from Protein (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

45. RNA and protein extraction from the same tissue (Ivan J Delgado)

46. SDS Removal Guard Columns

47. Small Scale MBP-fusion Protein Purification (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

48. Subcellular Fractionation of Proteins (Bowtell Lab Manual, Peter MacCallum Cancer Institute)

49. Sucrose Density Gradient Fractionation of Yeast Membranes (Dohlman Lab)
Test Tube for Ion Exchange Chromatography (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

50. The Protein Purification Facility (Dr. Mario Lebendiker)

51. Tips for acid elution of peptides (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

52. Whole Cell Protein Extracts (Morrisey Lab, University of Pennsylvania)
This is for both cytoplasmic and nuclear proteins.
Protein Purification - How to Purify Proteins.

Suggested Reading

Protein Purification Handbook What is Biotechnology?Protein Engineering Methods What is a Protein?What are Enzymes?

Databases of Virology

2010-01-15T21:33:00.000-08:00

The Journal of Virology Online - http://jvi.asm.org/

Online version of the Journal of Virology, the premier journal for peer-reviewed papers on virology research.

Retrovirology - http://www.retrovirology.com
A peer reviewed journal that publishes reviews and original research on retroviruses.

Virus Databases Online - http://www.ictvdb.rothamsted.ac.uk/
A comprehensive index of viruses provided by the International Committee on Taxonomy of Viruses. Also provides a picture gallery, identification tool and software tools.

All the Virology on the WWW - http://www.virology.net/
Links to virology information, collection of virus pictures, virology bookshop, jobs, and course notes.

UK Clinical Virology Network - http://www.clinical-virology.org
News, clinical tests, and current viruses in circulation.

HIV Sequence Database - http://www.hiv.lanl.gov/content/index
Collection HIV and related genetic sequence data and computer analyses - from the Los Alamos Natl. Lab.

American Society for Virology - http://www.asv.org/
A forum for investigators of human, animal, insect, plant, fungal and bacterial viruses in areas such as clinical, ecological, biological or biochemical research.

Paramyxoviruses - http://en.wikipedia.org/wiki/Paramyxovirus
Information from Wikipedia on these negative-sense single-stranded RNA viruses, responsible for a number of human and animal diseases, including their taxonomy, morphology and pathogenesis.

Poxvirus Bioinformatics Resource - http://www.poxvirus.org
The Poxvirus Bioinformatics Resource Center has been established to provide resources to the scientific community for basic research and to facilitate the development of novel antiviral therapies and vaccines against human orthopoxvirus infections as well as approaches for environmental detection of virions and the rapid diagnosis of disease.

Viral Replication - http://en.wikipedia.org/wiki/Viral_replication
Information from Wikipedia on the attachment, entry, and expression of viral genomic material in host cells.

Wadsworth Center - New York State Department of Health - http://www.wadsworth.org/databank/viruses.htm
Information and images regarding; Rabies virus, Herpes virus, Enteroviruses, Norwalk virus, Human Parvovirus B19 and Rotavirus.

Virology Down Under - http://www.uq.edu.au/vdu/
An Australian based portal and educational site providing links and information on human viruses and related areas of science.

Hidden Killers--Deadly Viruses - http://library.thinkquest.org/23054/gather/index.shtml
Basics about viruses, our bodies' immune systems, information about specific viruses, and how these 'hidden killers' could potentially be used as bio-weapons. There are also weekly/bi-weekly feature articles on various facets that involve viruses.

A New Germ Theory - http://www.theatlantic.com/issues/99feb/germs.htm
The dictates of evolution virtually demand that the causes of some of humanity's chronic and most baffling "noninfectious" illnesses will turn out to be pathogens -- that is the radical view of a prominent evolutionary biologist.

EVA: European Virus Archive - http://www.european-virus-archive.com
Project aiming to create a European network of high calibre laboratories with the expertise to collect, amplify, characterise, standardise, authenticate, distribute and track all viruses.

Viper: Virus Particle Explorer - http://mmtsb.org/viper/
Descriptions of icosahedral virus capsid structures, along with structural and computational analysis.

VirHostNet - http://pbildb1.univ-lyon1.fr/virhostnet
Knowledgebase for virus-host and protein-protein interaction networks.

National Collection of Pathogenic Viruses - http://www.ukncc.co.uk/html/members/ncpv/ncpv.htm
NCPV preserves well-characterised, authenticated human pathogenic viruses in a secure facility. The agents or nucleic acids derived from these viruses are supplied to the worldwide scientific community according to national and international guidelines.

Baculovirus.com - http://www.baculovirus.com
Technical knowledge, information, newsgroup, helpful in using the Baculovirus expression vector system and insect cell expression.

Virology - http://www.elsevier.com/locate/issn/0042-6822
This journal publishes the results of basic research in all branches of virology, including the viruses of vertebrates and invertebrates, plants, bacteria, and yeasts/fungi.

What the Heck is a Virus? - http://people.ku.edu/~jbrown/virus.html
Provides a relatively easy to understand answer to this question.

Virusys Corporation - http://www.virusys.com
Specialists in the design and manufacture of virus related antibodies and antigens. A full range of laboratory services is available that includes infectious dose determination, viral protein purification and custom antibody production.

Blogging for Bacteriophages - http://www.phagehunter.org/
Blog by Tim Sampson who takes a look at recent developments in the world of microbiology.

The Molecules of HIV - http://www.mcld.co.uk/hiv/
The life cycle, immune response, and schematic diagram of the human immuno-deficiency virus.

Plant Virology - http://www.bioinvision.dk/plantvir1.html
Introduction to virus structure, transmission, and transport.

The Phage Phorum - http://www.phages.org
A place to find general information regarding biophages and an open discussion of topics related to biophages.

Veterinary Virology - http://www.pitt.edu/~super1/lecture/lec3351/001.htm
Lecture presentation on the replication of viruses within animals cells.

Viral Replication - http://www.epidemic.org/theFacts/viruses/viralReplication.html
Description of the hepatitis virus replication process.

Medical Virology - http://www.medicalvirology.com
Scientific findings, clinical therapies research, and public health aspects on viruses of medical importance.

GeneJohnson Inc. - http://www.genejohnson.net
HIV genomic data storage and analysis systems. HIVBase allows one to organize HIV sequence data, perform complex statistical analysis, and collaborate data all in one program.

Viralman - http://viralman.110mb.com
Educational site for students provided by H.Niazmand at Azad University of Kazeroun, with downloads and an image gallery.

Hepadnavirus Testing, Inc. - http://www.hvtinc.com

The site describes the services provided by Hepadnavirus Testing, Inc., which uses HBV and DHBV to evaluate disinfectants, inactivation processes, and antiviral drugs for the biotechnology and pharmaceutical industries.

Antiviral Intelistrat - http://www.antiviralintelistrat.com/
Database of antiviral drugs and vaccines. It regroups over 1700 product files containing detailed information on compounds and vaccines related to the prevention or treatment of viral diseases. Subscription needed.

Chicken Flu: Recombinant Genes on the Loose - http://www.synapses.co.uk/science/fluvirus.html
Information regarding viral genes, replication, and strain evolution. Written in 1997 but updated in 2005.

Seminars in Virology - http://www.sciencedirect.com/science?_ob=JournalURL&_cdi=7150&_auth=y&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=7ea28456e36b7099a712a2f126c0c294
Review journal dedicated to keeping scientists informed of developments in the field of virology on a topic by topic basis.

Virus Ultrastructure - http://web.uct.ac.za/depts/mmi/stannard/linda.html
This page is designed primarily as an introductory manual for students of virology,but anyone who is interested is invited to take a brief "peep" at some of the inhabitants of the microbial Lilliput Land.

Journal of General Virology - http://www.sgm.ac.uk/JGVDirect/
Archive of downloadable PDF virus journals.

(Source: Google directory)

Protocols in Virology

2010-01-15T21:31:00.000-08:00

Creation and Use of Infectious Virus Vector

http://www.stewartlab.net/Protocols/Making_Virus_1...

(Stewart Lab, Washington University at St. Louis)

Extraction of Geminiviral DNA from a Highly Mucilaginous Plant Protocol http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb18/R00-056.pdf

Protocol for the extraction of geminiviral DNA from a highly mucilaginous plant (Abelmoschus esculentus). Includes: Extraction; Alkali lysis; Cloning.

Generation of recombinant baculoviruses by co-transfection (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

http://www.its.caltech.edu/%7Ebjorker/Baculovirus-...
For co-transfection prepare at least 10mg of highly purified plasmid DNA. Spodoptera frugiperda cells are sensitive to some contaminants found in crude plasmid preparations, which cannot be removed by phenol/chloroform extraction or ethanol precipitation.

Gradient Purification of AT2 Inactivated HIV-1 Protocol http://ubik.microbiol.washington.edu/protocols/bl3/at2.htm

Protocol for gradient purification of AT2 inactivated HIV-1.

HIV-1 p24 Antigen Capture ELISA Protocol http://ubik.microbiol.washington.edu/protocols/bl3/p24elisa.htm

Protocol for HIV-1 p24 antigen capture ELISA.

Information Resources for Adjuvants and Antibody Production

http://www.nal.usda.gov/awic/pubs/antibody/
Article explaining the function of adjuvants, providing guidelines for their use, particularly Freund's adjuvants, and introducing some alternatives that are commercially available.

Immunohistochemistry: In Situ Hybridization

http://www.immunoportal.com/
Includes details of immunoenzyme techniques, immunofluorescence and in situ hybridizations, with a photograph gallery and discussion board.

Purification of Bacteriophage lambda Arms: Centrifugation through Sucrose Density Gradients Protocol

http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3990

Protocol is used to prepare the arms of any bacteriophage {lambda} vector.

Purification of Bacteriophage lambda Particles by Isopycnic Centrifugation through CsCl Gradients

http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3968

Isopycnic centrifugation through CsCl gradients is used to prepare infectious bacteriophage {lambda} particles of the highest purity that are essentially free of contaminating bacterial nucleic acids.

Purification of Recombinant Adeno-Associated Virus (rAAV) and Parovirus Protocol http://www.axis-shield.com/densityhome/optiprep/M04.pdf

Protocol for the purification of recombinant adeno-associated virus (rAAV) and parvovirus in pre-formed iodixanol gradients.

Retroviral infection of primary human keratinocytes (PHKs)

http://www.methodbook.net/cellcult/infectn.html

(Matt Lewis, Department of Pathology, University of Liverpool)

SDS-PAGE Sample Preparation of Adenovirus Infected Cells Protocol - http://www.bio.com/protocolstools/protocol.jhtml?id=p1752

Protocol for SDS-PAGE sample preparation of adenovirus infected cells.

TEV Purification Protocol http://openwetware.org/wiki/Keating:Experimental_Protocols:TEV_purification

Protocol for TEV purification.

Titration of Retrovirus Protocol

http://www.methodbook.net/virus/titratn.html

Protocol for the titration of retrovirus.

Viral Detection and Concentration in Food http://members.ift.org/NR/rdonlyres/69B323CE-B14C-4DD6-8F56-B25898C9EFB0/0/crfsfsv01n2p082085ms20010312.pdf

Methods and information related to viral detection and concentration in food. Includes: Nucleic acid amplification methods; Recent progress in polymerase chain reaction detection; Detection of human enteric viruses in non-shellfish food; Choice of primers for the detection of human caliciviruses in foods; Biosensors and microarray detection; Alternative confirmation methods.

Virion RNA Dot Blot Protocol

http://www.bio.com/protocolstools/protocol.jhtml?id=p617

Protocol describes the production of a dot blot membrane spotted with RNA isolated from virus particles.

Virus detection protocols for west nile virus in vertebrate and ...

www.ncbi.nlm.nih.gov/pubmed/12904372
Protocols to detect the presence of WNV have been refined since 1999 for ... Polymerase Chain Reaction/methods; RNA, Viral/isolation & purification ...

Virological Laboratory Techniques & Resources

(Source: All the Virology on the WWW)

BioGuide - PCR - general information about the polymerase chain reaction including primer design, hot-start, etc.

BioProtocol, Inc. - This site contains a large collection of high quality protocols for the life science researcher. These protocols include offerings from the virology field of study. The company has a highly reputable editorial board to oversee the quality of its protocol collection as well as an editorial staff committed to procuring and producing the highest quality protocols available on-line.

Detailed protocol for gold labeling proteins for electron microscopy. This protocol describes a modified antibody-protein A-gold technique that eliminates the aggregation problem that frequently occurs with the usual procedure.

Cell Biology Laboratory Manual by Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College, St. Peter, MN.

SPOTTER: a new tool to measure viral plaques. The plaque assay is a classical virological technique, originally developed for bacteriophages by Felix d'Herelle (1917). Dulbecco and Vogt subsequently adapted the assay for mammalian viruses, and it is still widely used for virus isolation and purification, and to determine viral titers. The plaque assay is also frequently used to study in vitro growth properties of mutant viruses. A freeware program,called SPOTTER, operating under Win95 and up, allows convenient and precise analysis of large numbers of plaques.

Protocol On-Line - an exhaustive collection of methods and protocols for molecular and cellular biology from academic and comercial laboratories. There is also a Q&A sections that may be helpful for solving lab technique problems.

Comprehensive Protocol Collection - This resource attempts to offer a comprehensive collection of molecular biology protocols. The site links to protocols either maintained directly by the Ambros Lab at Dartmouth or at other locations around the world.

The HHMI Virtual Lab: This interactive laboratory, developed with Shockwave, demonstrates how an enzyme-linked immunosorbent assay (ELISA) is carried out and some of the experimental problems that may be encountered. A free Shockwave plugin is required.

Molecular Biology Protocols - a General Resource from the NOAA of Molecular Methods, Manuals, and some great Links.

PCR in situ: New Technologies with Single Cell Resolution for the Detection and Investigation of Viral Latency and Persistance" from the University of Minnesota

PCR Jump Station - This site attempts to provide scientists with a comprehensive directory for the polymerase chain reaction (PCR) and related techniques.

Labexplorer.com is a new web site written by clinical laboratorians for clinical laboratorians. Designed to be a comprehensive resource for those interested in clinical laboratory practices, the site provides a mechanism of communication between laboratorians, access to experts in a variety of clinical fields, job and career information, and comprehensive links to laboratory sites, diagnostic companies, organizations & interesting topics.

Molecular Biology Protocols on the Web

a great collection of protocol links from the publishers of Molecular Biology Today.

Review Articles on Embryonic Stem Cell Research (3)

2010-01-15T21:30:00.002-08:00

67.

The transcriptional foundation of pluripotency.

Chambers I, Tomlinson SR.

Development. 2009 Jul;136(14):2311-22. Review.PMID: 19542351 [PubMed - indexed for MEDLINE]Related articles Free article

68.

When stem cells meet immunoregulation.

Tasso R, Pennesi G.

Int Immunopharmacol. 2009 May;9(5):596-8. Epub 2009 Jan 29. Review.PMID: 19539568 [PubMed - indexed for MEDLINE]Related articles

69.

Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo.

Zernicka-Goetz M, Morris SA, Bruce AW.

Nat Rev Genet. 2009 Jul;10(7):467-77. Review.PMID: 19536196 [PubMed - indexed for MEDLINE]Related articles

70.

Gene regulatory networks in embryonic stem cells and brain development.

Ghosh D, Yan X, Tian Q.

Birth Defects Res C Embryo Today. 2009 Jun;87(2):182-91. Review.PMID: 19530135 [PubMed - indexed for MEDLINE]Related articles

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The relationship between pluripotency and mitochondrial DNA proliferation during early embryo development and embryonic stem cell differentiation.

Facucho-Oliveira JM, St John JC.

Stem Cell Rev. 2009 Jun;5(2):140-58. Epub 2009 Apr 3. Review.PMID: 19521804 [PubMed - indexed for MEDLINE]Related articles

72.

An evaluation regarding the current situation of stem cell studies in Turkey.

Arda B, Aciduman A.

Stem Cell Rev. 2009 Jun;5(2):130-4. Epub 2009 May 12. Review.PMID: 19521803 [PubMed - indexed for MEDLINE]Related articles

73.

Policy interoperability in stem cell research: demystifying harmonization.

Isasi RM.

Stem Cell Rev. 2009 Jun;5(2):108-15. Epub 2009 Mar 31. Review.PMID: 19521802 [PubMed - indexed for MEDLINE]Related articles

74.

Diversity in public views toward stem cell sources and policies.

Einsiedel E, Premji S, Geransar R, Orton NC, Thavaratnam T, Bennett LK.

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75.

Emerging ethical, legal and social issues associated with stem cell research & and the current role of the moral status of the embryo.

Zarzeczny A, Caulfield T.

Stem Cell Rev. 2009 Jun;5(2):96-101. Epub 2009 May 14. Review.PMID: 19521800 [PubMed - indexed for MEDLINE]Related articles

76.

The stem cell research environment: a patchwork of patchworks.

Caulfield T, Zarzeczny A, McCormick J, Bubela T, Critchley C, Einsiedel E, Galipeau J, Harmon S, Huynh M, Hyun I, Illes J, Isasi R, Joly Y, Laurie G, Lomax G, Longstaff H, McDonald M, Murdoch C, Ogbogu U, Owen-Smith J, Pattinson S, Premji S, von Tigerstrom B, Winickoff DE.

Stem Cell Rev. 2009 Jun;5(2):82-8. Epub 2009 May 27. Review.PMID: 19521798 [PubMed - indexed for MEDLINE]Related articles

77.

New perspectives in human stem cell therapeutic research.

Trounson A.

BMC Med. 2009 Jun 11;7:29. Review.PMID: 19519878 [PubMed - indexed for MEDLINE]Related articles Free article

78.

GABA's control of stem and cancer cell proliferation in adult neural and peripheral niches.

Young SZ, Bordey A.

Physiology (Bethesda). 2009 Jun;24:171-85. Review.PMID: 19509127 [PubMed - indexed for MEDLINE]Related articles

79.

Large-scale mouse mutagenesis.

Cartwright EJ.

Methods Mol Biol. 2009;561:275-83. Review.PMID: 19504078 [PubMed - indexed for MEDLINE]Related articles

80.

Naive and primed pluripotent states.

Nichols J, Smith A.

Cell Stem Cell. 2009 Jun 5;4(6):487-92. Review.PMID: 19497275 [PubMed - indexed for MEDLINE]Related articles

81.

Embryonic stem cell microRNAs: defining factors in induced pluripotent (iPS) and cancer (CSC) stem cells?

Gunaratne PH.

Curr Stem Cell Res Ther. 2009 Sep;4(3):168-77. Review.PMID: 19492978 [PubMed - indexed for MEDLINE]Related articles

82.

Adult stem cells and mammalian epimorphic regeneration-insights from studying annual renewal of deer antlers.

Li C, Yang F, Sheppard A.

Curr Stem Cell Res Ther. 2009 Sep;4(3):237-51. Review.PMID: 19492976 [PubMed - indexed for MEDLINE]Related articles

83.

Hematopoietic cell development in the zebrafish embryo.

Bertrand JY, Traver D.

Curr Opin Hematol. 2009 Jul;16(4):243-8. Review.PMID: 19491671 [PubMed - indexed for MEDLINE]Related articles

84.

Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.

Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA.

J Clin Invest. 2009 Jun;119(6):1438-49. doi: 10.1172/JCI38019. Epub 2009 Jun 1. Review.PMID: 19487820 [PubMed - indexed for MEDLINE]Related articles Free article

85.

The basics of epithelial-mesenchymal transition.

Kalluri R, Weinberg RA.

J Clin Invest. 2009 Jun;119(6):1420-8. doi: 10.1172/JCI39104. Review.PMID: 19487818 [PubMed - indexed for MEDLINE]Related articles Free article

86.

EMT: when epithelial cells decide to become mesenchymal-like cells.

Kalluri R.

J Clin Invest. 2009 Jun;119(6):1417-9. doi: 10.1172/JCI39675. Review.PMID: 19487817 [PubMed - indexed for MEDLINE]Related articles Free article

87.

Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs.

Kashyap V, Rezende NC, Scotland KB, Shaffer SM, Persson JL, Gudas LJ, Mongan NP.

Stem Cells Dev. 2009 Sep;18(7):1093-108. Review.PMID: 19480567 [PubMed - indexed for MEDLINE]Related articles

88.

MicroRNA and stem cell regulation.

Wang Y, Russell I, Chen C.

Curr Opin Mol Ther. 2009 Jun;11(3):292-8. Review.PMID: 19479662 [PubMed - indexed for MEDLINE]Related articles

89.

Role of ubiquitin ligases in neural stem and progenitor cells.

Naujokat C.

Arch Immunol Ther Exp (Warsz). 2009 May-Jun;57(3):177-88. Epub 2009 May 29. Review.PMID: 19479207 [PubMed - indexed for MEDLINE]Related articles

90.

Pericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell niche.

Díaz-Flores L, Gutiérrez R, Madrid JF, Varela H, Valladares F, Acosta E, Martín-Vasallo P, Díaz-Flores L Jr.

Histol Histopathol. 2009 Jul;24(7):909-69. Review.PMID: 19475537 [PubMed - indexed for MEDLINE]Related articles

91.

Genetic regulation of proliferation/differentiation characteristics of neural progenitor cells in the developing neocortex.

Mitsuhashi T, Takahashi T.

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92.

Stem cells for epidermal melanocytes--a challenge for students of dermatopathology.

Cramer SF.

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93.

Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation.

Snykers S, Henkens T, De Rop E, Vinken M, Fraczek J, De Kock J, De Prins E, Geerts A, Rogiers V, Vanhaecke T.

J Hepatol. 2009 Jul;51(1):187-211. Epub 2009 Apr 2. Review.PMID: 19457566 [PubMed - indexed for MEDLINE]Related articles

94.

Amniotic stem cells for cellular cardiomyoplasty: promises and premises.

Walther G, Gekas J, Bertrand OF.

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95.

Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells.

Butler JT, Hall LL, Smith KP, Lawrence JB.

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96.

Prospects for pluripotent stem cell-derived cardiomyocytes in cardiac cell therapy and as disease models.

Freund C, Mummery CL.

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97.

Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review.

Mizuno H.

J Nippon Med Sch. 2009 Apr;76(2):56-66. Review.PMID: 19443990 [PubMed - indexed for MEDLINE]Related articles Free article

98.

Pluripotent stem cells.

Verfaillie C.

Transfus Clin Biol. 2009 May;16(2):65-9. Epub 2009 May 12. Review.PMID: 19442552 [PubMed - indexed for MEDLINE]Related articles

99.

Stem cell research and therapy for liver disease.

Navarro-Alvarez N, Soto-Gutierrez A, Kobayashi N.

Curr Stem Cell Res Ther. 2009 May;4(2):141-6. Review.PMID: 19442198 [PubMed - indexed for MEDLINE]Related articles

100.

Adult stem cells as an alternative source of multipotential (pluripotential) cells in regenerative medicine.

Kuçi S, Kuçi Z, Latifi-Pupovci H, Niethammer D, Handgretinger R, Schumm M, Bruchelt G, Bader P, Klingebiel T.

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