For cryosections many antibodies work well without antigen retrieval step as they are either not fixed or fixed for relatively short period of time, so that the croslinking did not occur in a significant manner. Presence of  detergent in blocking buffer and antibody diluent is enough to ensure good antibody penetration. If your staining doesn't work you can play with the percentage of Tween that you are using. If antigen retrieval is needed you can try enzymatic methods., such as proteinase K treatment. Just remember that you need to tightly control incubation times to get good, repetitive results with this approach.

Good luck.

I incubate 5 minutes in Methanol.

The first step is to try not using antigen retrieval (AR).  AR is usually only needed for fixed tissue, to break up the cross linkages formed through fixation, especially formalin.  Minimal fixation of the cryosections is recommended, once mounted on slides, to maintain the tissue section integrity during staining.

Dear Mohammed,

Thanks for your suggestion. This human and rat nerve are my control tissue, fixed more than a month. I used this to optimize my GFP-positive tissue (also fixed for 1 week), in frozen section. I admit my mistake as the sample being fixed quite long in time. I did tried the method that you suggested, looks like it is not working, I didnt observed any expression. Yes, the tissues were preservedand not destroyed but why I didnt get any positive tissue. Previously, I omitted AR step, but also it wasnt worked well.

Hi Agniezka, thanks for your suggestion. Is it may work in case of fixation in long period of time? 

Dear Christin,

Thanks. What concentration would you suggest for me? I will do try so. Thanks

Hi Philip. Thanks for your suggestion. But, in case of overfixation, is there any alternative to improve the expression if someone did a mistake like this? Or there is a way for it? Thanks

For your information, actually I did optimized these 2 antibodies, on paraffin section S100B and MPZ, both worked well after heated AR and withour heated AR respectively. But then, on frozen section I faced a problem when the heater AR step destroyed my section. So I would to know any method that you had tried before or whether anyone had faced the same problem before. Thanks a lot!  

Khairunnisa, I apologize.  I missed the part where you stated that the tissue was fixed (over-fixed) first and then cryosections taken from them.  Are you using charged or silanized slides to promote adhesion of the tissue to the slide?  Over-fixed tissue, giving no expression, usually requires aggressive AR such as boiling citrate.  You can shorten the time in citrate solution to see if tissues survive, you can try EDTA instead of citrate, you can try different pH's of citrate or EDTA.  It is a tricky thing to find what works, and every antigen is a little different. I just thought of something else.  If the tissue is already fixed, maybe you can continue to process it and embed it in paraffin.  Paraffin sections will survive antigen retrieval much better.

The enzymatic digestion should work for the crosslinks as it brakes proteins into smaller pieces. The important thing is to keep it mild enough to keep your antigen intact. For this you should test multiple enzyme concentrations and incubation times.

100% Methanol ;-)

Dear Philip, no worries! I would love to try as soon as possible. But, as you suggested, I cant prepare a paraffin block for it as I already sectioned my GFP-positive nerve tissue (suggested to preserve GFP by doing frozen block not paraffin) and I need to optimize on frozen section. So then I can co-stain my GFP-expressed cells with nerve marker to see whether my transplanted cells incorporate in nerve regenaration or not. 

Dear Agnieszka, will also do try ASAP. Thanks so much! 

Thanks Christin, will do try. Thanks!

Oh, you want to stain GFP positive tissue. The methanol could kill the GFP fluorescence. Than try to stain it without fixation first. After optional fixation you can also detect GFP per antibody staining as mentioned in that link: https://www.researchgate.net/post/GFP_fluorescence_after_fixation10

I remember my former colleague had an similar problem. But I do not remember how he solved it. You can ask him on researchgate: Andrii Domanskyi

Dear Christin,

Actually my GFP-tissue already fixed in 4% PFA. As we did intracardiac perfusion using 4% PFA, then we harvested the nerve graft, I did fixed in 4% PFA. I hope that I can optimize the ABs on frozen section followed by on my sample. Thanks, I will check on that link and your friend. Thank you so much!

I think that fixing the GFP will probably kill the fluorescence. I would try anti-GFP staining, as  Christin Geißler mentioned. We sometimes have problems seeing GFP fluorescence even in unfixed tissue

I believe that harsh antigen retrieval is generally only needed for paraffin embedded sections. I would try to incubate tissue sections with 1% Triton X-100 in PBS/TBS for 5-30 min at room temp before staining. Sometimes this helps.

Dear Paul, 

Luckily we managed to observe the GFP expression under fluorescence microscope. But the challenge is to co-stain with my nerve markers to confirm whether the cells do incorporate or not. Like you suggested to incubate in Triton X, is it for overfixation tissue or normal unfixed frozen section? I tried to incubate my overfixated tissue in SDS 1% , but no expression observed while after heat AR, I managed to observe the expression but the tissue destroyed. I will try to do so as suggested. Thanks

It  probably causes  certain level of  denaturation  of your nerve   marker   under chemical treatment.   Could you try using different fluorescent  dye to label the marker of interest ?     

Normally with frozen sections you would not require antigen retrieval . The purpose of antigen retrieval is to break the aldehyde bonds that form when you fix the tissue .  So in the case of Frozen sections they would not generally require antigen retrieval. Antigen retrieval on unfixed tissue will destroys  the section. You may want to try using 2 different fluorescent dyes and doing IMF instead on frozen tissue

Dear Galayanee,

Dear Galayanee, I did fixed in 4% PFA only, no other vigorous chemical treatment, maybe it does give effect, not sure. I will try with other Ab. Thanks

Dear Hasiena, as mentioned, we fixed before we do frozen section. After I do heat AR, managed to observe the expression on my control sample but my tissue was 80% destroyed. Maybe I need to reduce the incubation as others had suggested. What's your opinion? and what is IMF? Thanks

Dear Khairunnisa

What pH are you using for your antigen retrieval. The higher the pH the greater the possibility of the tissue washing off  or being destroyed. You could look at using enzymes like trypsin and pepsin to expose your antigen.IMF this is immunofluorescence which works well on frozen tissue. A fluorochrome is attached to the antibody and the final end product viewed under a fluorescence microscope. When you fix with PFA , how long are you fixing for and how big is your tissue?. From what you mentioned your control tissue seems to be working fine. Have a look to see if there is any difference in the length of time your specimens were in fixative compared to your control. Your  problem  may be with fixation.

Dear

I perform Ihc on fresh muscle biopsies with several antibodies but it does not require any antigen retieval methods ...directy I apply interested antibody ..then secondary antibody followed by chromogen the counter stain ...the results are perfect...good luck for you

Dear all, thanks for kind response, I would like to share that I managed to observe immunoexpression after I did antigen retrieval step using the heated method in citrate buffer under an optimized incubation time  in 100C of citrate buffer. It really helps. Thanks a lot!