Avidin biotinylated horseradish peroxidase complex abc hrp

To optimize immunohistochemical staining testis sections, both commercially available control (Zyagen, San Diego, CA, USA) and LCT sections were immersed in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven (2 × 5 min, 700 W). After overnight incubation at 4 °C with primary antibodies, (they are listed in Table 2) respective biotinylated antibodies (anti-rabbit and anti-mouse IgGs; 1: 400; Vector, Burlingame CA, USA) and avidin-biotinylated horseradish peroxidase complex (ABC/HRP; 1:100; Dako, Glostrup, Denmark) were applied in succession. Bound antibody was visualized with 3,3′-diaminobenzidine (DAB) (0.05%; v/v; Sigma-Aldrich) as a chromogenic substrate. Control sections included omission of primary antibody and substitution by irrelevant IgG (Bilinska et al. 2018 ).

Testicular tissues were cut into 4-µm-thin sections. For antigen retrieval, endogenous peroxidase neutralization and blocking of non-specific binding sites were performed as described previously [80 (link)]. Thereafter, the sections were incubated overnight at 4 °C with a primary antibody: anti-GPER (Abcam; Cambridge UK; ref. no.39742), anti-INSL3 (Santa Cruz Biotechnology; Dallas, TX, USA ref. no sc-134587), anti-PKA II Antibody (Santa Cruz Biotechnology; Dallas, TX, USA; ref.no. sc-908), or anti-IP3 I (Sigma-Aldrich Saint Louis, MO, USA; ref.no. BT AP04603). Next, biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and avidin-biotinylated horseradish peroxidase complex (ABC/HRP; Dako, Glostrup, Denmark) were applied in succession. The staining was developed using 3,3′-diaminobenzidine (DAB). Positive controls and negative controls in the absence of primary antibodies were performed for each immunostaining. Thereafter, sections were slightly counterstained with Mayer’s hematoxylin and mounted using DPX mounting media (Sigma-Aldrich; Saint Louis, MO, USA). Serial sections were examined with a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany). The experiments were repeated three times on sections obtained from different animals.