Subratab,

You are using very harsh retrieval for something that should not need any at
all. Methacarn fixative is not crosslinking, therefore you are destroying
your tissue by proteolytic enzymes and heat. Even tissue that has been fixed
with formalin, but not long enough, produces diminished nuclear staining.
Try your method without any retrieval, when fixed in methacarn.

Katri

Katri Tuomala
St.Joseph's Healthcare
Hamilton, Ontario, Canada


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


> 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).
Tissue
> 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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