Lsm510 exiter

Immuno-staining was performed as described [21] (link). Briefly, cells grown on coverglasses were washed with PBSCM (PBS containing 1 mM CaCl2 and 1 mM MgCl2) and then fixed with 3% paraformaldehyde in PBSCM at 4°C. After sequential washing with PBSCM supplemented with 50 mM NH4Cl, cells were permeabilized with 0.1% saponin (Sigma, St. Louis, MO, USA) in PBSCM for 15 min at room temperature, and were subjected for immuno-staining using the antibodies indicated, then labeled withTexas red-, FITC- or Cy5 conjugated secondary antibodies. Immuno-labeled cells or/and GFP-expressing cells were analysed under the laser scanning confocal immunofluorescence microscope (Carl Zeiss LSM510 EXITER, Zeiss, Jena, Germany).

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.