Re: [Histonet] Explain the biochemical basis of 'no counterstain with hematoxylin'

From:" Katri Tuomala"

One more thing I want to add to my previous message, Subratab.  In your IHC
protocol, you use alkaline phosphatase conjugated secondary antibody. You
don't need the 3% H2O2 in methanol step, which is to block endogenous
peroxidase. That is also too harsh for alcohol fixed tissue.


----- Original Message ----- 
From: "Subratab" 
Sent: Sunday, October 26, 2003 2:53 PM
Subject: [Histonet] Explain the biochemical basis of 'no counterstain with

> Dear all
> I am staining (IHC) rat renal tissue for ED1. When I am using
> Methacarn-fixed tissue I am not getting any counterstain with hematoxylin.
> Nuclear positions are showing blank space (ghost-like rounded gaps).
> is showing just-yellowish appearance.
> I have stained the methacarn-fixed slides with hematoxylin without going
> through my IHC protocol (deparaffinization and hematoxylin). And the
> staining is OK. So the methacarn-fixed tissue has no problem with
> hematoxylin. It indicates that my IHC protocol is not campatible with
> hematoxylin stain when I am using methacarn-fixed tissue.
> On the otherhand, when I am using paraformaldehyde fixed tissue with the
> same protocol, I am facing no problem to counterstain with hematoxylin.
> 1. Can you please explain the biochemical basis? 2. Can the problem be
> solved by methyl green counterstaining?
> I am expecting clarification and suggestion from the histonet
> experts.Following is my IHC protocol:
> Deparaffinization and rehydration
> Digestion of protein cross-links by trypsin
> Microwave exposure in citrate buffer
> Blocking with 1% non-fat milk
> Primary Ab (ED1) diluted in PBS (1% BSA + 0.3% Triton x100)
> 3% H2O2 in methanol
> AP conjugated polymer (anti-mouse Ig)
> Fast red with substrate buffer and levamisol (from Dako)
> Counterstaining with hematoxylin and mounting with permafluor.
> Washing solution is PBS. (ED1 staining is OK. The problem is with
> counterstaining)
> Subrata Biswas.
> University of Campinas, brazil.
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