2.4 Immunofluorescence (IF) staining
The sperm from patients and the normal controls and mouse sperm cells were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 10 min, and blocked with 5% BSA or 30% donkey serum for 60 min at room temperature. The slides were then sequentially incubated with primary antibodies at 4°C overnight. The primary antibodies used were anti-CFAP47 (1:50) and α-tubulin (1:100, A11126, Thermo Fisher Scientific). The next day, 1 × PBS was used to wash the samples three times. Then, the samples were incubated with AlexaFluor 594 anti-rabbit secondary antibodies (1:1000, 1927937, Thermo Fisher) and AlexaFluor 488 anti-mouse secondary antibodies (1:1000; A32723, Thermo Fisher) for 2 h at room temperature or coincubated with peanut agglutinin (PNA, 1:50, RL‐1072‐5, Vector), CFAP65 (1:50, HPA055156, Sigma–Aldrich), CFAP69 (1:50, bs-15278R-A647, Bioss), COXⅣ (1:50, 11242-1-AP, Proteintech) and SEPTIN4 (1:50, 12476-1-AP, Proteintech). Subsequently, 1 × PBS was used to wash the slides three times. Then, the slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI, D9542‐1MG, Sigma‐Aldrich) to label the nuclei. Finally, the slides were sealed in coverslips. Images were obtained by a laser scanning confocal microscope (Olympus). For immunofluorescence staining of mouse and human testes, after careful xylene dewaxing and gradient ethanol rehydration, the tissue sections were submerged in boiling 10 mM citrate buffer (pH 6.0) for 10 min. Then, the sections were cooled to room temperature and washed with 1 × PBS for 5 min. Subsequently, the sections were treated with 3% hydrogen peroxide solution for 10 min. After washing with 1× PBS, the slides were blocked with 10% normal donkey serum for 30 min and incubated with primary antibodies at 4°C overnight and with Alexa Fluor 488 or Alexa Fluor 594 antibodies for an additional 2 h at room temperature. The primary antibodies used were anti-CFAP47 (1:50), anti-CFAP65 (1:50) and anti-CFAP69 (1:50). Slides of testicular tissues were observed using an LSM800 confocal microscope (Carl Zeiss AG).