Immunohistochemical Analysis of Kisspeptin, Androgen Receptor, and Inflammatory Markers

Paraffin-prepared sections were stained using the avidin–biotin peroxidase system for the Kisspeptin, androgen receptor, TNFα, Nrf2, and Ki67 detection using a primary antibody (rabbit polyclonal antibody). Following three PBS washes (5 min each), the sections were incubated for 30 min at room temperature with biotinylated secondary antibody and avidin–biotin complex (Vectastain^® ABC-peroxidase kit, Vector Laboratories, Burlingame, CA). The color was developed using 3,3′ -diaminobenzidine (DAB) substrate (Vector^® DAB, Vector Laboratories). Mayer’s hematoxylin was used as a counterstain. The stained sections were examined by light microscopy (LEICA ICC50 W) in the Image Analysis Unit of the Human Anatomy and Embryology Department, Zagazig University (Khamis et al., 2023 (link)).