Sc 1511

Western blot analysis was performed using the protein extracts from ELISA quantifications. Aliquots of each sample containing equal amounts of protein (30 μg) were subjected to SDS-polyacrylamide gel electrophoresis, and β-actin was used as loading control. Gels were transblotted onto a polyvinylidene difluoride membrane, and the blots were blocked in 3% skim-milk for 1 h at room temperature, followed by incubation overnight at 4 °C with a primary goat polyclonal antibody directed to ICAM-1 (1:200 dilution; sc-1511; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or with primary rabbit polyclonal antibody directed to iNOS (1:200 dilution; sc-8310; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GFAP (1:200 dilution; sc-9065; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Finally, blots were incubated for one hour at room temperature with HRP-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed with Clarity Western enhanced chemiluminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Images were acquired (ChemiDoc XRS⁺; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the optical density (OD) of the bands was evaluated (Image Lab 3.0 software, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The data were normalized to the corresponding OD of β-actin. All experiments were performed in duplicate.

Western blot analysis was performed using the protein samples from ELISA quantifications. Aliquots of each sample containing equal amounts of protein (30 μg) were subjected to SDS-PAGE, and β-actin was used as loading control. Gels were transblotted onto a PVDF membrane, and the blots were blocked in 3% skim-milk for 1 h at room temperature, followed by incubation overnight at 4 °C with a primary goat polyclonal antibody directed to ICAM-1 (1:200 dilution; sc-1511; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or with primary rabbit polyclonal antibody directed to GFAP (1:200 dilution; sc-9065; Santa Cruz Biotechnology) or iNOS (1:200 dilution; sc-8310; Santa Cruz Biotechnology). Finally, blots were incubated for one hour at room temperature with HRP-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology) and developed with Clarity Western enhanced chemiluminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Images were acquired (ChemiDoc XRS+; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the optical density (OD) of the bands was evaluated (Image Lab 3.0 software; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The data were normalized to the corresponding OD of β-actin. All experiments were performed in duplicate.

Immunohistochemical staining was performed as described previously (21). Briefly, the tissue sections were boiled in citrate buffer (Dako target retrieval solution, S1699, DAKO, Carpenteria, CA) to achieve antigen retrieval for 20 min. The slides were then incubated with peroxidase blocking solution (S2023, DAKO) for 15 min and protein block serum-free (X0909, DAKO) for 30 min. Tissue sections were incubated overnight at 4 C with primary antibodies: type I collagen (1:200; 1310-01; Southern Biotech, Birmingham, AL, USA), intercellular adhesion molecule-1 (ICAM-1) (1:200; sc1511; Santa Cruz Biotechnology, Santa Cruz, CA, USA), F4/80 (1:200; 14-4801-81; eBioscience, San Diego, CA, USA) and fibroblast-specific protein 1 (FSP-1)/S100A4 (1:200; ab41532; Abcam, Cambridge, UK). Second antibodies were incubated for 30 min at room temperature and then treated with horseradish peroxidase-conjugated streptavidin (P0397, DAKO) for 30 min. To visualize the immunocomplexes, the testicular sections were treated with 3-amino-9-ethyl carbazole substrate solution (K3464, DAKO). The F4/80-positive macrophages and FSP1-positive fibroblasts per high-power field were counted at a magnification of $\times$ 200. The ImageJ software was used to measure the fibrotic areas of type I collagen and ICAM-1 positive area in 10 randomly chosen non-overlapping fields at a magnification of $\times$ 200.