Re: [Histonet] double immuno staining

From:Gayle Callis

It probably is your fixation which is compatible with one antibody but not
the other. Formalin is the culprit and you still "lose" antigenicity due to
cross linking of antigen by the formaldehyde and paraformaldehyde.  You may
want to try perfusion with PLP (something one lab does in our department)
then post fix in that fixative. Snap freezing of fresh tissue DOES preserve
antigens, so you can try unfixed tissue, snap frozen, then fixed with other
fixative AFTER you cut overnight RT air dried frozen sections.  If you use
acetone, 4C for 10 min or another fixative choice, a fixation panel,
antigen retrieval is not needed, and you may have far more success with
both antibodies.  

Antigen retrieval and enzyme digestion (also using strong peroxidase
blocking - chews sections off slides at times) methods on frozen sections
can damage morphology, so you may want to work with a fixative where you
never have to do any retrieval whatsoever.    

Chris van der Loos may respond to this message also and he is the master of
multiple staining.  

At 04:19 PM 5/5/2004 +0200, you wrote:
>Hi folks,
>I need some help.
>I am struggling with a double immunostaining for somatostatin receptor and
>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
>Histonet mailing list
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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