Mab65591

Triplicate for each rabbit of sections of ovarian tissue and GCS-crawling film were used for immunofluorescence staining. Ovarian tissue sections and GCS-crawling film were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min. Then cell-climbing slices were incubated with an antifollicle-stimulating hormone receptor (MAB65591, 1: 200, R&D Systems, Minneapolis, United States) and ovarian sections were incubated with anti-CTSB (48118-1, 1:300, Signalway Antibody, Maryland, United States) rabbit polyclonal antibody for 1 h at 37°C. The sections were then incubated with allophycoerythrin (APC)-goat antirabbit Ig G (GB25303, 1:400, Servicebio, Wuhan, China) at 37°C for 45 min. Finally, the ovarian section and cell-climbing slices were taken out and mounted with DAPI medium (Solarbio). The results were observed under a laser scanning confocal microscope (Nikon Eclipse C1).

FSHR (follicle stimulating hormone receptor) protein is usually used as a marker protein to identify GCS. The cells were seeded on culture plates plated with cell-climbing slices. After reaching 70% confluence, cells were fixed with 4% paraformaldehyde and then permeated with 0.1% Triton X-100. For immunohistochemical staining, the cells were incubated with anti-FSHR (MAB65591, 1: 200, R&D Systems, Minneapolis, MN, USA) for 1 h at 37 °C, and then incubated with goat anti-rabbit IgG (bs-0296G-FITC, 1:400, Bioss, Beijing, China) at 37 °C for 45 min. Finally, the cells were taken out and mounted with DAPI (4′,6-Diamidino-2-phenylindole, Dihydrochloride) medium (Solarbio, Beijing, China). The cells were then observed and imaged under a fluorescence microscope (Nikon Eclipse C1, Tokyo, Japan).