Hello! Does anyone know a good protocol to perform immunohistochemistry on the skin of mice tail?
Thank you very much in advance :)
I have used the protocol found in the supplemental materials of the attached paper to stain mouse tail. I hesitate to comment because we have an autostainer here, so much of the protocol is done with a machine, and we stained the tissue for fluorescent markers, not brightfield like usual IHC, so please feel free to disregard if this protocol is not what you are looking for. It is optimized for the skin of mouse tail, however.
Protocol: Antigen retrieval in paraffin sections was carried out by boiling for 15 min in Citrate buffer (pH 6). Permeabilisation was performed using 0.5% Triton X 100 (Sigma) and non-specific antibody binding was blocked using PBS containing 3% BSA and 10% bovine serum. Incubations with primary and fluorescent secondary antibodies were carried out for 2h at room temperature. Slides were mounted using Pro-Long Gold anti-fade reagent (Invitrogen) containing the nuclear counterstain 4′,6-diamidino-2-phenylindole (DAPI). To prepare wholemounts, skin was incubated in PBS/5 mM EDTA in a 37 °C water bath for 1 to 4 h. The epidermis was then separated from the dermis and fixed with 2% formaldehyde for 10 minutes. Antibodies were diluted in PB buffer (PBScontaining 0.5% milk powder, 0.25% fish skin gelatin and 0.25% Triton X 100) and incubations carried out overnight. GFP immunohistochemistry was performed with an automatic staining system (Leica ST5020, Leica Microsystems; Wetzlar, Germany) and the Bond Intense R Detection kit (Leica Microsystems; Wetzlar, Germany).
Best of luck in your experiments.
Thank you very much for your reply and for the paper.
This is very useful for my experiments, I really appreciate your help.
Good luck to you too :)