Periodic acid schiff and hematoxylin

Human testicular tissue fragments were fixed with 4% PFA overnight, paraffin-embedded and sectioned (5 μm). The tissue slides were de-paraffinized, rehydrated, incubated for 30 minutes in sodium citrate buffer (10 mM sodium citrate, pH 6.0, 0.05% Tween-20) for antigen retrieval. The tissue was then incubated in peroxidase block for 10 minutes and washed in PBS and blocked with a buffer containing 3% bovine serum albumin and 5% normal goat serum. Subsequently, sections were stained for 90 minutes at room temperature with rabbit anti-UCHL1 (1:1000, 7863-0507, Biogenesis). Isotype matched normal IgG was used as negative control. Primary antibody was detected using goat anti-rabbit HRP conjugated secondary antibody (1:200, sc-2054, Santa Cruz Biotechnology) for 30 minutes. Metal enhanced DAB substrate kit was used to detect staining (Thermo Scientific). The tissue was then counterstained with Periodic acid-Schiff and hematoxylin (Sigma-Aldrich).

Sectioned tissue slides were deparaffinized, rehydrated and incubated antigen retrieval buffer as mentioned above. Slides were incubated in peroxidase block for 10minutes, washed in PBS and blocked with goat blocking buffer containing 3% bovine serum albumin and 5% normal goat serum (Jackson ImmunoResearch) then stained with primary antibodies or negative control (refer to antibody table). Goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody for 30 minutes. DAB substrate was used to detect staining, sectioned were counterstained with periodic acid-Schiff and hematoxylin (Sigma-Aldrich).