Immunohistochemical Analysis of Testicular Apoptosis

IHC was performed according to a previously reported method (Li et al., 2015 (link)). Briefly, testicular sections were dewaxed with xylene and dehydrated in serially graded ethanol solutions. The endogenous peroxidase activity was quenched with 3% H₂O₂ in distilled water. The sections were applied with anti-Bcl-2 antibody (ab196495, Abcam Inc., Cambridge, MA, USA; 1:50), anti-Cleaved caspase-3 antibody (AB3623, Merck Millipore, Billerica, MA, USA; 1:20), and anti-Cytochrome C (CytC) antibody (ab13575, Abcam Inc., Cambridge, MA, USA; 1:100) separately and then incubated overnight at 4°C. As a negative control, some sections were reacted with PBS instead of the specific antibodies. After reacting with a biotin-conjugated secondary antibody (ab6720, Abcam Inc., Cambridge, MA, USA; 1:1,000) for 1 h at room temperature, the sections were stained using the FAST DAB Peroxidase Substrate (Sigma, St Louis, MO, USA) and counterstained with haematoxylin for 10 s. The slides were then dehydrated and mounted for observation.