since one year now I am trying to do different stainings for 6 receptor subtypes in rat brain.
The antibodies I use are from different companies, four polyclonals and two monoclonals.
The aim of my work was or is, to obtain a characteristic distribution pattern for the different receptor subtypes in
specific rat brain areas.
Although it took me really a lot of time to get the stainings work in PFA fixed tissue, I finally managed to get an intense staining
with antigen retrieval using citrate buffer with pH6 in a steamer.
During my analysis I was very surprised because all the stainings using different antibodies look completely the same. I tried to
do costainings and even there it appeared that the different receptor subtypes seemed to be localized in the same cells which was
very surprising for us.
I switched now to a completely different receptor using an antibody that worked fine without antigen retrieval.
But since I have to do costainings with the first mentioned antibodies I had to do the AR.
So what should I say, I got exactly the same staining pattern again.
If I omit the primary antibody I get no staining at all but no matter if I use IF or ICH, I get the same distribution of positive cells
for all 7 antibodies tested so far.
This now is no longer only surprising, this cannot be real anymore. There are two opportunities, but one
is more likely. I will be the first person publishing a new phenomenon in nature or
there is something completely wrong with my staining.
Is there anybody who has an idea or is aware of the problem? Can this be due to the AR with citrate buffer?
At the moment I wonder if I can use any of my results analysed so far