Benzidine-peroxidase staining for blood (Conlon lab)

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Protocol submitted by VGP from Conlon lab protocols [1]

link to protocol document [2]

Benzidine Staining

(Hemoglobin histochemical stain)

1) Make up 4X Benzidine stock

100 mg Benzidine
3.125 ml HAc (Glacial)
3.125 ml H2O
store @ 4°C in the DARK

2) Fix Embryos in 1X benzidine for 5-10 minutes

500ul Benzidine/1480ul H2O

3) Add 20ul 30% H2O2

4) Stain 2-15 minutes

5) Wash several times in 12% Glacial Acetic Acid

6) Fix 1.5 h in MEMFA

7) Change MEMFA 3X

8) Wash 3-4X EtOH

9) Store @ 4°C or 20°C in EtOH