Re: [Histonet] more PAS on ISH

From:John Kiernan wrote:
> I started my alcian PAS protocole, the alcian blue (1g /100ml in 3% glacial
> acetic acid) doesn't penetrate the emulsion and color the tissue but eats
> it away the emulsion (the silver grain ISH signals with it). Are there any
> alternative alcian blue protocols????

That's surprising. Is the emulsion fixed and hardened?
You need to use a combined fixer-hardener containing
alum as well as Na2S2O3. We use the same general
purpose Kodak fixer that's used for films and papers.
Na2S2O3 alone will fix (remove unexposed silver
halides) but will not harden the emulsion. Hardening
is similar to fixing in the histological sense. The
Al3+ ions from the alum cross-link gelatin molecules
of the emulsion. 

The alcian blue solution you describe, in 3% acetic
acid, should have a pH of 2.5. In your earlier email
you said pH 4. 
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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