Clusterin

The purity of the primary Sertoli cells was evaluated with Sertoli cell-specific protein markers using the immunofluorescence approach (Bernardino, Alves et al. 2018). The cultured Sertoli cells were fixed with 4% paraformaldehyde (Cat# BM-155, Boston Bioproducts, Ashland, MA) at room temperature for 30 min and washed with PBS twice. Cells were then permeabilized with 0.5% Triton X-100 in PBS solution for 20 min at room temperature and blocked with a blocking buffer containing 3% Bovine serum albumin (BSA)/ 0.2% Triton X-100 in PBS for 1.5 h at room temperature. The cells were incubated with the primary antibodies' dilution in 1%BSA/.05 % Tween-20 /PBS solution containing SOX9 (Cat# ab26414, Abcam, Waltham, MA) or GATA-4 (Cat# ab84593, Waltham, MA) or Clusterin (Cat# ab69644, Abcam, Waltham, MAUK) at 4°C overnight. After washing with 0.05 % Tween-20 PBS buffer, the cells were incubated with secondary antibodies conjugated to differential fluorochromes DyLight-488 (Cat# 35552, Thermo Fisher Scientific, Waltham, MA). And the nuclei were stained with Hoechst 33342 (Cat# H3570, Thermo Fisher Scientific, Waltham, MA) in 1% BSA and 0.05% Tween-20 in PBS for 1.5h at room temperature. In addition, Alexa Fluor 488 Phalloidin (Cat# 8878S, Cell Signaling, Boston, MA) was used for F-actin cytoskeleton staining by incubating with cells for 30 min at room temperature.