[Histonet] Antibody staining on non-pfa fixed tissue

From:"Gregory A. O'Sullivan"

Hello HistoNetters,

I realise that this may be a question that has arisen on previous occasions, however, I have been unable
to find an exact answer from a search of previous email correspondence on HistoNet.

I would like to detect and examine the distribution of my protein of interest in mouse brain sections
(with particular emphasis on the hippocampus) and compare it to other known markers.
The problem is that the antibody I am using is extremely sensitive to pfa fixation but the marker antigens
are best detected on fixed tissue. I have tried to overcome this problem by fixing cryostat sections
briefly with pfa before using different antigen retrieval protocols (either microwave or heated bath incubation
in Citrate Bufer) to unmask epitopes but this results in a staining that is suboptimal for both antigens
(protein of interest and marker).

One 'idea' would be to try and incubate these dried crystat section with my antibody of interest and then post fix
with pfa before proceeding with the marker antibody incubation. My concern here is that these unfixed sections
will undergo degradation prior to fixation but perhaps someone might have experience with regard to this.
An alternative, I have come across from previous emails, is to fix the tissue by dipping the sections in acetone
for 1 to 2 minutes at rtp. I was wondering whether anyone has any experience of using such a protocol for antibodies that
are pfa fixation sensitive?

Anyhow, any suggestions would be extremely helpful.


Gregory A. O'Sullivan PhD
MPI for Brain Research
Deutschordenstrasse 46
60528 Frankfurt
Tel: +49 69 96769315
Fax: +49 69 96769441
Mob: +49 163 6359371
email: osullivan@mpih-frankfurt.mpg.de
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