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| A protocol for chemical detection of heme containing proteins, useful for detecting blood in wholemounts. Submitted by Jerry Thomsen [http://www.xenbase.org/community/person.do?method=display&personId=760&tabId=0].
| | '''Antibodies''' |
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| Benzidine is a reagent that forms a blue precipitate upon oxidation by the heme group of hemoglobin in the presence of hydrogen peroxide. Thus it serves as a histochemical stain specific for differentiated red blood cells. The figure is from A. Hemmatti-Brivanlou and G.H. Thomsen (1995). Ventral mesodermal patterning in Xenopus embryos: expression patterns and actvities of BMP-2 and BMP-4. Devl. Genet. 17: 78-89. [http://www.xenbase.org/literature/article.do?method=display&articleId=20388]
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| '''Staining protocol:'''
| | [[Monoclonal 3G8- pronephros and otic vesicle]] |
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| Fix embryos or tissues for 5 minutes in 12% glacial acetic acid containing 0.4% benzidine (Sigma # B-3503 CARCINOGEN).
| | [[Monoclonal 4A6 - pronephros]] |
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| Start reaction by addition of hydrogen peroxide to a final concentration of 0.3%. Incubate at RT (22 deg C). Color should develop within 10-15 minutes.
| | [[antibody template]] |
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| Alternatively, the specimens can be incubated in an acetic acid/benzidine solution already containing freshly added hydrogen peroxide.
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| Monitor reaction for color development and photograph immediately.
| | '''Lectins''' |
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| The blue precipitate is unstable in the presence of peroxide, and the peroxide will also gradually destroy the specimens.
| | ECL - stains pronephric nephrons |
| | | FPA - stains cement gland and scattered epidermal cells |
| Peroxide can be washed out with 12% acetic acid and fixed in methanol to stablize the precipitate, but the color will turn brown and lose intensity.
| | FWGA - stains cement and hatching glands |
| | | FVVL - stains cement gland and (weakly) scattered epidermal cells |
| Protocol is adapted from Orkin S.H., Harosi, F.L. and Leder (1975): Differentiation of erythroleukemic cells and their somatic hybrids.
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| PNAS 72:98-102.[http://www.pnas.org/content/72/1/98.abstract]
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| [[File:Bst50vent.jpg]]
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Revision as of 16:11, 12 January 2010