[Histonet] double immuno staining

[Veronique Andriessen]

Thanks everyone for all the kind replies!

I am definately gonna try the PLP fixation. I would love to use acetone
fixation, but this is not possible for the somatostatin receptor. It will
destroy most of the cell membranes.

As for antigen retrieval, I've tried HIER at pH 6 and 9 and EIER with
pronase and trypsin at different time intervals, dilutions and temperatures.
10 minutes at RT with trypsin-EDTA gives the best result (morphology vs
staining intensity) for this fixation.

I am also going to look for another desmin antibody clone to mix with the
one I'm already using. Hopefuly this will enhance the signal.


Sincerely yours,

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


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Veronique
Andriessen
Sent: woensdag 5 mei 2004 16:19
To: Histonet
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



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