Author Topic: Different antibodies - same staining pattern after AR  (Read 5296 times)

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Offline tanja08

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Different antibodies - same staining pattern after AR
« on: July 23, 2009, 05:42:44 AM »
Hi everyone,

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  ???.
Greeting
Tanja



Different antibodies - same staining pattern after AR
« on: July 23, 2009, 05:42:44 AM »

Offline Cardio

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Re: Different antibodies - same staining pattern after AR
« Reply #1 on: July 24, 2009, 10:26:52 AM »
Quote
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.

Could you elaborate on the above. Did you expect a different staining pattern when using this antibody. I am assuming this was your postive control that did not give you the expected results.

Others can better answer the AR question but some things I would consider if I were you.

First I would try frozen sections to see if it is the AR.
Second I would check the sequence the ab's are against. Since you are dealing with receptor subtypes is it conserved or not between the receptors (long shot but worth looking into).

Good luck.

Offline tanja08

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Re: Different antibodies - same staining pattern after AR
« Reply #2 on: July 27, 2009, 10:26:09 AM »
Hi,
you are right. I expected a different staining pattern using this antibody.
So Friday I compared the staining pattern with AR and without and they look completely different.So at the moment I wonder what is real. As far as I understand the AR should only damask epitopes that were hidden due to the PFA fixation. So it sounds plausible that there are more positive cells. But for me it is somehow funny that all antibodies used show the same staining pattern after AR. But what might be the explored thing where all antibodies bind to?

Checking the sequences is a good point, I will do so.

Re: Different antibodies - same staining pattern after AR
« Reply #2 on: July 27, 2009, 10:26:09 AM »