Ns02l

Immunostaining of the spermatozoa was performed as described previously (Sha, Xu, et al., 2017). Briefly, the prepared spermatozoa were smeared onto poly‐L‐lysine coated slides, allowed to air‐dry, washed in phosphate‐buffered saline (PBS), fixed in 4% PFA (F8775, Sigma, United States) for 10 min at RT, and washed twice in PBS, followed by permeabilization with 0.2% Triton X‐100 (93443, Sigma, USA). Then, the samples were blocked with PBS containing 1% bovine serum albumin (A1933, Sigma, USA) and 2% normal goat serum (NS02L, Millipore, USA) for 30 min at RT. Slides were incubated with rabbit anti‐EIF4G1 (15704‐1‐AP, Proteintech, USA), rabbit anti‐COXIV (11242‐1‐AP, Proteintech, USA), or rabbit anti‐ATP6 (55313‐1‐AP, Proteintech, USA) primary antibodies and costained with anti‐acetylated tubulin (66200‐1‐Ig, Proteintech, USA) primary antibodies 1 hr at RT, followed by incubation with Alexa Fluor^® 594‐conjugated goat anti‐rabbit IgG (ZF‐0516, zsbio, China) and Alexa Fluor^® 488‐conjugated goat anti‐mouse IgG (ZF‐0512, zsbio, China) secondary antibodies for 45 min at RT. Slides were subsequently washed three times in PBS, mounted with Vectashield containing DAPI (Vector Laboratories) and examined under a laser scanning confocal immunofluorescence microscope (LSM510 Exiter, Carl Zeiss, Germany). The specific antibodies used in this assay are listed in Table S2.