Re: Methyl Green pyronin staining

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From:Laurence Reilly <>
To:"Sarah Christo" <>, <>
Date:Thu, 29 Jul 1999 10:34:34 +1000
Content-Type:text/plain; charset="us-ascii"

Dear Sarah,
         The method we have used came originally from Elias,J.M. (1969)
Stain Technology 44  201 via Arthur Rowlatt at CSIRO in Melbourne, Australia.

Like all good chefs we have altered the method to suit our own needs, which
has been to demonstrate plasma cells.

Carnoy's is the fixative of choice but formalin fixed tissues can be
stained if the ratio of the staining solutions is altered.

First, the 2% Methyl Green solution must be cleared of violet colours by
extraction with chloroform
1. Take sections to water
2. Stain in Methyl Green-Pyronin solution (freshly made)      3 hrs

If the fixative was formalin                 If the fixative was Carnoy's
2% aq. Pyronin Y     1.5ml                     2% aq. Pyronin Y    3ml
2% aq. Methyl Green  3.0ml                     2% aq. Methyl Green 3ml
Tap Water to 50ml                              Tap Water to 50ml
Townsville tap water is "about neutral"--distilled water dosen't work.
(Buffer solutions may be required)

3. Rinse with tert-butanol
4. Dehydrate in tert-butanol
5. Clear in xylene and mount in DPX (or similar)

Nuclei   --   green-purple
Plasma cell cytoplasm    -    red

Another reference that I have been given for formalin fixed tissues using a
pH 3.8 is Ahlquist, J. (1972) Stain Technology 47  17, but I haven't tried

Good luck with it.     Regards,    Laurie.

Mr.Laurie Reilly                          Ph 07 4781 4468
Physiology & Pharmacology                 Fax  07 4781 5558 
Aust.Inst.of Tropical Vet.& Animal Sc.    
James Cook University           
       Townsville  Qld. 4811                            

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