[Histonet] RE: double immuno staining

[C.M. van der Loos]

Dear Veronique,
I am wondering if it would be possible to embed the specimens after the perfusion into paraffin. This would make your tissue morphology more "resistant" to pretreatment procedures. Since you have it so optimally fixed by perfusion one may wonder what can go wrong with the antigens during full paraffin embedding? 
I am a bit surprised about killing the staining of desmin. Usually this is a good and stable antigen that cannot leak away or so. Perhaps testing different clones of anti-desmin is helpful to you. 
Hope this helps,

Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center
Amsterdam - The Netherlands

----- Original Message ----- 
>From  "Veronique Andriessen" <vandries <@t> vub.ac.be> 
Date  Wed, 5 May 2004 16:19:08 +0200 
To  "Histonet" <histonet <@t> lists.utsouthwestern.edu> 
Subject  [Histonet] double immuno staining 
Hi folks,

I need some help.

I am struggling with a double immunostaining for somatostatin receptor and
desmin.

All my previous double stainings worked beautifully, but they were performed
on fresh frozen acetone fixed pancreatic tissue.

For staining of somatostatin receptor I have to fix with formaldehyde. I use
20 minutes 0.75% PFA  perfused tissue which is snap frozen embedded in
tissue freezing medium and cut on a cryostat. I was advised to do it this
way to prevent loss of antigenicity.
Morphology is quite good.
This fixation works for the somatostatin receptor staining, but it kills the
desmin signal.
I was able to retrieve most of the desmin signal by EIER for 10 minutes with
trypsin-EDTA, but now the morphology is awful.

Can someone give me some advice?

Sincerely yours,

Veronique Andriessen BAS
Lab. Molecular Liver Cell Biology,
Free University Brussels (VUB)Belgium