Immunohistochemical Staining of CD31 and FSHR

In order to expose the epitopes for the IHC procedure, the deparaffinized and rehydrated sections were boiled in Target Retrieval Solution (DakoCytomation, Glostrup, Denmark, S2369) in a microwave oven (twice 700 W for 5 min). Once cooled and washed with PBS, the endogenous peroxidase was blocked by using a 3% solution of perhydrol in methanol, and then the slides were incubated overnight at 4 °C with primary antibodies: monoclonal anti-CD31 (abcam, Cambridge, UK, ab231436; final dilution 8 μg/mL) and polyclonal anti-FSHR (abcam, Cambridge, UK, ab150557; final dilution 1:50) in antibody diluent with background-reducing components (Dako, Santa Clara, CA, USA, S3022). To visualize the antigen–antibody complexes, a Dako LSAB+System-HRP was used (DakoCytomation, Glostrup, Denmark, K0679) based on the reaction of avidin–biotin–horseradish peroxidase with DAB as a chromogen according to the included staining procedure instructions. Sections were washed in distilled H₂O and counterstained with hematoxylin. For the negative control, specimens were processed in the absence of primary antibodies. Positive staining was determined microscopically (Leica DM5000B, Wetzlar, Germany) through visual identification of brown pigmentation [23 (link)].