Immnol fluorence staining secondary antibody dilution buffer

Cells were fixed in 4% paraformaldehyde, sealed with Immnol Fluorence Staining Secondary Antibody Dilution Buffer (Beyotime), and then incubated with a 1:200 dilution of ZO-1 antibody (ab96587, Abcam) at 4°C for 24 h. After washing, cells were incubated in a 1:200 dilution of FITC-labeled goat anti-rabbit IgG (H+L) (Beyotime) for 30 min at 37°C. DAPI was prepared for nuclei staining at a 1:1000 dilution for 5 min. Images were captured with confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Cell fluorescence was analyzed by Image J software (Rawak Software, Inc. Germany).

CCs were seeded into 4-well plates (Nunc, Thermo Fisher Scientific Life Sciences, Massachusetts, USA) at a density of 1.2 × 10⁵ cells per well and oocytes were cultured in 4-well plates before analysis. Both CCs and oocytes were washed three times with cold PBS (Sigma, Billerica, USA) and fixed in 2% PFA for 20 min. After washing three times with Triton X-100 containing Immunol Staining Wash Buffer (Beyotime Biotechnology, CN), fixed samples were stained for 90 min at room temperature by 6 μl SYBR-labeled mouse-anti-human ChemR23 antibody (PA550932, Thermo Fisher Scientific Life Sciences, Massachusetts, USA) diluted with 300 μl Immnol Fluorence Staining Secondary Antibody Dilution Buffer (Beyotime Biotechnology, CN). Cells were washed again with Immunol Staining Wash Buffer for three times. The anti-fluorescence-quenching agent with DAPI (6-diamidino-2-phenylindole; DAPI Fluoromount-G TM; Yeasen Biotechnology, CN) was used to label nuclei. Images of CCs were captured with a fluorescence microscope (NE900, Nexcope, USA) in plates. For oocytes in plates, cells were gently shifted to the glass slide, slipcovered and scanned by the same microscope.