Ab5535

HuPMCs in 24-well plates were fixed in 4% PFA for 1 hr, then rinsed and stored in PBS for later immunofluorescence staining. Retinal vibratome sections and huPMCs were initially blocked in 5% normal goat serum (Sigma) overnight at 4°C. Sections were immunostained with primary antibodies: PHGDH (Millipore, ABS571, 1:1000), CRALBP (Abcam, ab15051, 1:500), GFAP (Abcam, ab53554, 1:1000), Carbonic Anhydrase II (Abcam, ab6621, 1:1000), SOX9 (Millipore, ab5535, 1:1000) diluted in PBS containing 1% normal goat serum and 0.5% Triton X-100. Vibratome sections were incubated for 4 nights at 4°C. Thereafter, huPMCs were incubated with species-specific secondary antibodies conjugated with Alexa Fluor 488 (green) or 594 (red) (Molecular Probes) at a 1:1000 dilution for 2 hr at room temperature; while the vibratome sections were incubated at 4°C overnight. Cells and sections were then rinsed with PBS and nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific). After mounting in VECTASHIELD antifade mounting medium (Vector Laboratories), immunofluorescence labelling of huPMCs and vibratome sections was captured with the ZEISS confocal laser-scanning microscope (LSM700, Carl Zeiss) and ZEN Blue software.

Immunoblot and immunofluorescence analysis were performed as previously described18 (link). Primary antibodies used were: SOX9 (AB5535, Millipore), phospho-SOX9 (ab59252, abcam), BMI1 (05-637, Millipore), E-cadherin (BD610181, BD Transduction Laboratories), Vimentin (M7020, DAKO), N-Cadherin (BD610920, BD Transduction Laboratories), phospho-S6 Ribosomal protein (Cell Signaling Technology^®, #4858) and β-actin (AC-15, Sigma). For Western blot detection of primary antibodies, we used HRP-linked antibodies (Santa Cruz Biotechnology) and detection was performed by chemiluminescence using NOVEX ECL Chemi Substrate (ThermoFisher). For immunofluorescence, secondary antibodies conjugated with fluorochromes were used and nuclear DNA was stained with Hoechst 33342 (Sigma). Images were obtained at a 40x magnification.

Test samples from control and all-dosed-CORT mice were embedded with Tissue-Tek OCT compound, frozen in liquid nitrogen, and stored at − 80 °C until examination. Embedded testes were dissected with a cryostat at 5 m. The sections were fixed with acetone for 2 min at − 20 °C. For immunohistochemistry, separate sections were incubated with primary antibodies: rabbit anti-p27 polyclonal antibody (ab190851, Abcam; 1:100 dilution) that recognizes CDK inhibitor, rat anti-GATA-1 monoclonal antibody (N6) (sc-265, Santa Cruz Biotechnology, Inc.; 1:100 dilution) or rabbit anti-SOX-9 polyclonal antibody (ab5535, Millipore; 1:2000 dilution), that recognizes a marker of Sertoli cells. Following overnight reaction, sections were washed and incubated with a biotinylated anti-rabbit or rat immunoglobulin IgG (1:200) for 30 min and labeled with avidin-biotinylated horseradish peroxidase (PK-6101, Vectastain ABC Elite Kit, Vector Laboratories, Burlingame, CA, United States) for 30 min at room temperature. Following washing, immunoreactivities were visualized with 3,3′-diaminobenzidine (DAB), and sections were counterstained with Gill’s hematoxylin III. Twenty circular seminiferous tubules were randomly selected for each animal, and immunoreactive cells were counted using the ImageJ 1.51J8 program.