Benzidine-peroxidase staining for blood (Thomsen lab)

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A protocol for chemical detection of heme containing proteins, useful for detecting blood in wholemounts. Submitted by Jerry Thomsen [1]. 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. [2]

Staining protocol:

Fix embryos or tissues for 5 minutes in 12% glacial acetic acid containing 0.4% benzidine (Sigma # B-3503 CARCINOGEN). Start reaction by addition of hydrogen peroxide to a final concentration of 0.3%. Incubate at RT (22 o C). Color should develop within 10-15 minutes. Alternatively, the specimens can be incubated in an acetic acid/benzidine solution already containing freshly added hydrogen peroxide. Monitor reaction for color development and photograph immediately. The blue precipitate is unstable in the presence of peroxide, and the peroxide will also gradually destroy the specimens. 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. Our protocol is adapted from Orkin S.H., Harosi, F.L. and Leder (1975): Differentiation of erythroleukemic cells and their somatic hybrids. PNAS 72:98-102.[3]