Dear Jan,

I worked with frozen section 20um thick brain which was fixed with immersion method, and the brains  were preparing daily. I didnt get good staining with fixed stored brains. Unfortunately dont have  useful experience to share.I would be glad to help. 

Hallo Jan, if you have already mount your sections on superfrost plus slides and dried them out over night, you can try  an antigen retrievel in Citrate buffer pH 6 pr EDTA buffer pH 9 in the microwave. If not you can woke with free floating sections. I recommand 0,2 % Triton X-100 in PBS or TBS as a delution medium, over night incubation of the primary antibody at room temperature.  If you don't get any positiv rsults after you have applied the flouorophore labelled secondary antibody You can try it with biotinylated secondary antibody and Strepavidin-Fluorophore. This technique is much more sensitive then the 2-step methode. With Streptavidin you have the possibility to enhance the result  with anti-Streptavidine and a scond application of the same Streptavidin-fluorophore.  Please run a negative control (no primary antibody) to be able to distinguish between autofluorescence and other unspeciic background staining. I hope, this will help you.

Hi Jan,

Do you have free-floating or slide-mounted tissues?

For the first ones, I use to wash the tissues in PBS, an incubate with 0.1M citrate buffer (pH 6.0) 30 minutes at 50 degrees. Then wash again in PBS, and tissues are ready for IHQ protocol.

For slide-mounted tissues, my experience is not very good. But when I used to work with parafin-embebed tissues, I used a pressure cooker for 5 minutes in citrate buffer, the problem was that tissues were a little damaged after.

There's a really good protocol paper for antigen retrieval that could help you.

https://www.ncbi.nlm.nih.gov/pubmed/10634500

Hope this helps...

Hi, Manoji,

Thanks for sharing your protocol. How did you do autoclaving at 105 degree for 1 minute? Do you use decloacking chamber? Thanks