Benzidine-peroxidase staining for blood (Thomsen lab)

The staining protocol is:

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.