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

[Subratab]

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.