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).