Immunofluorescence Analysis of CFAP251 in Sperm

The immunofluorescence test was carried out according to a protocol described previously (Wang et al., 2021 (link)). The sperm were washed three times with PBS, smeared onto glass slides and air-dried. The sperm slides were next fixed with 4% formaldehyde in PBS at room temperature for 20 min, followed by washing in PBS three times. Slides were then blocked in 5% bovine serum albumin (A8020; Solarbio, China) and incubated overnight at 4°C with primary antibody anti-CFAP251 (H00144406-B01P, Abnova, China) and HRP-conjugated secondary antibody (GB23301, Servicebio, China). Furthermore, incubation was carried out at room temperature for 10 min with Cy3-tyramide (G1223, Servicebio, China), followed by washing with PBS three times. Then, the slides were incubated with anti-α-tubulin antibodies (GB12200, Servicebio, China) and HRP-conjugated secondary antibody followed by FITC-tyramide (G1222, Servicebio, China). The slides were counterstained with 5 mg/ml DAPI (G1012, Servicebio, China) and mounted with mounting media (G1401, Servicebio, China). Finally, fluorescence images were taken using a confocal microscope (Nikon Eclipse CI, Nikon, Japan).