I have samples of brain tissues which are over fixed due to prolonged exposure with paraformaldehyde. What can I do for getting appropriate signals in immunohistochemistry on these paraffin embedded tissues ?
Did you try to stain and obtained no signal or unspecific signal? Some targets are OK with over-fixation and you might be lucky.
Antigen-retrieval protocols are pretty common (Heat, acid, enzymatic see link below) but it might not work for all the targets.
You will have to tweak the conditions for every target you want to stain.
http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol
Did you try to stain and obtained no signal or unspecific signal? Some targets are OK with over-fixation and you might be lucky.
Antigen-retrieval protocols are pretty common (Heat, acid, enzymatic see link below) but it might not work for all the targets.
You will have to tweak the conditions for every target you want to stain.
http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol
we had the sample which was fixed for a year in PFA4% but it was still ok,
for having the best quality especially in the brain it's better first minimize background which you can prolong Antigen retrieval with sodium citrate and make sure that each time made a fresh sodium citrate and check PH to be at 6, the another important point is that choose proper detergent based on ur antibodies between triton and tween. since u might get much unspecific background staining to rid of that use 10% serum with 1% BSA plus ur primary antibody in 0.03%TBST this will give u highest specific binding, also DAB time should be adjusted 10s to 1min sometimes 2min based on tissue types. the last point is that keep ur unstained slides in the 4 degrees fridge, in that case, they won't lose their sensitivity to the antibody.
gook luck.
Masoumeh
Hi Myryam,
In our lab we have used samples that have been fixed for 1-2 year and they worked fine. We didn't need to process this samples differently. What we did was to fast a few samples fist just to be sure, but we had extra samples to do that.
we just did the normal Ethanol gradient and paraffin and that was all.
Hi, Myryam ,
I too agree with Erika as even, I tried with Ethanol gradient and it has worked well.
Thank You
Hi Maryam,
As mentioned before in the answers the stability of the epitope you research a) and the specificity of your antibody to the epitope b) will be your limiting factors. Otherwise I don't see an issue. The prolonged exposure to aldehydes leads to the so called "overmasking" of the epitope or to complete fragmentation of the protein of interest and therefore loss of the epitope/protein during processing and/or retrieval.
Just keep these possibilities in mind.
With best regards,
Ivan
Hi Maryam..
I agree with the reply by Masoume Majidi zolbin.. still please tell how long did you incubate with PFA? and was it made in PBS?
try your usual protocol or switch to citrate buffer (pH 6) for antigen retreival.. over night incubation with primary antibody, fresh reagents, with BSA in 2% TBS (both for blocking and antibody preparation) and good hands, you will be able to see the protein of interest.. If not.. kindly post again and we can discuss the issue..
All the best,
Ayesha
hi Maryam Borhani Haghighi ! I ahve a question. I'm having problems with the sectioning of over fixed brains. How did you managed the sectioning? Thank you very much!
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