Immpress hrp detection kit

Testes and epididymides were fixed in Bouin’s solution for 2 h at room temperature, embedded in paraffin, and sectioned at 5 µm thickness. Slides were de-waxed, rehydrated, and heated in 10 mM sodium citrate buffer (pH 6.0). The slides were then blocked with 2.5% horse serum (Vector Laboratories, California, USA) for 30 min, incubated with primary antibodies for 1 h, washed with PBS, incubated with the secondary antibodies (ImmPRESS-HRP detection kit, Vector Laboratories) for 30 min, and washed with PBS, at room temperature. The primary antibodies are as follows: anti-STRA8 (1:500 dilution; ab49405, Abcam, Massachusetts, USA), anti-BrdU (1:500 dilution; ab6326, Abcam), anti-SOX9 (1:200 dilution; AB5535, Millipore, Massachusetts, USA), and anti-γH2AX (1:200 dilution; ab11174, Abcam). For colorimetric detection, slides were developed using a DAB substrate kit (Vector Laboratories), counterstained with Mayer’s hematoxylin, dehydrated, and mounted with Permount (Fisher Scientific).

RISH was done by manual RNAscope Chromogenic assays, using RNAscope 2.5 HD Assay—BROWN or Duplex Assay according to the manufacturer (ACDBio). Chromogenic probes were Hs-IGF1, Hs-CXCL13, Hs-C7, Hs-CXCL13-C2, Hs-IGF1R-C2, Hs-SRD5A2-C2, and Hs-AR-C2. Semiquantitative scoring of RNAscope assays (based on dots per cell, 0–4 scale) was done per ACDBio guidelines. Immunohistochemistry was done using Tris-EDTA antigen retrieval, with ImmPRESS HRP Detection Kit and ImmPRESS Duet Double Staining Polymer Kit (HRP/AP) (Vector Laboratories). Primary antibodies were anti–Ki-67 (D2H10, 1:400) (Cell Signaling Technology), anti-CD20 (L26, 1:400) (MilliporeSigma), and anti-CD3 (MRQ-39, 1:800) (MilliporeSigma).