The benzidine method demonstrates the peroxidase-like
activity of haemoglobin. This can also be done with 
the leuco-patent blue method. The reduced (colourless)
dye is oxidized to its coloured form, an acid dye 
that then sticks to globin, which is a basic protein.

Fixation: formaldehyde, 48 hours maximum; paraffin sections.

Stock solution (leuco-patent blue):  To 100 ml of 1%
aqueous patent blue V (CI 42045, Acid blue 1) add 
10g zinc powder and 2 ml glacial acetic acid. 
Boil until pale yellow(about 10 minutes), cool and filter. 
Keeps 8 months. An aniline blue (soluble blue, methyl
blue etc; CI 42780) may be used instead of patent blue V.

Working solution: Stock solution 10 ml
                  Glacial acetic acid 2 ml
                  3% hydrogen peroxide 1 ml

Procedure:  Immerse hydrated sections in the working solution
for 3-5 minutes. Wash well in distilled water.
Counterstain if desired (eg 1% neutral red).
Wash, dehydrate, clear and cover.

Result: Haemoglobin (in red cells and, pathologically,
elsewhere) - blue. (Nuclei red if counterstained)

Reference: H.C.Cook 1974. Manual of Histological Demonstration
Techniques. London:Butterworths, p. 78-79. Cook cites Lison
(1938) and Dunn (1946). I haven't checked these references.
 
-- 
-------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan@uwo.ca
   http://publish.uwo.ca/~jkiernan/
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louise renton wrote:
> I am looking for a protocol for staining haemoglobin in FFPE* tissue. I
> recall that there is a technique that uses benzidine, but given the toxicity
> of this substance, i would prefer an alternative. Thank you in advance.
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