The tissue were perfusion-fixed with 4% PFA after PBS washing and the rat brains were remove and post-fixed for 48 hours.
Will it be necessary or not to allow antigen retrieval before proceeding with the immunohistochemistry?
Secondly, is the microwave-antigen retrieval heating method of the citrate buffer the best?
Yes, beautifully with material processed free floating. When in paraffin or frozen and thawed onto slides, epitope retrieval may be needed.
For epitope retrieval, there is not one way fits all. Some epitopes require alkaline treatment, others citrate is fine and still others are best at neutral pHs. I usually test the citrate along with the pH 8 method. See:
J Histochem Cytochem. 1995 Feb;43(2):193-201.
Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies.
Shi SR1, Imam SA, Young L, Cote RJ, Taylor CR.
Thank You Gloria.
My sections were PFA fixed and frozen sections were made. I was going to try a test run on slide mounted tissues.
The referred paper was for paraffin embedded tissues. Will it still apply for mine? Is epitope retrieval same as antigen retrieval?
Yes it does. You must conduct a titration of the antibodies (I would test 1:1000, 1:3000, 1:10,000 at least) so you will know the correct primary antibody concentration to use. I would NOT use fluorescence for this but
ABC elite staining with NiDAB as the substrate for peroxidase. Staining must be for 20 minutes. You may need to amplify the reaction, so contact me after you have done the basic titration. with mounted sections we keep the solutions on the slide by using a parafilm 'coverslip' to prevent drying out during incubation with antibodies and the sections should be in a moist chamber (over wet paper towels for example).
Hello Gloria, sorry I could not get back to you earlier concerning the titration on Iba-1. After trying it on different brain specimens (rats and mice) didn't have a positive effects So discouraged
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