2.4 Immunofluorescence staining
The spermatozoa samples were washed with Sperm Washing Medium (CooperSurgical Inc.) and then fixed onto slides with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 5% BSA. Next, the slides were sequentially incubated with primary antibodies (SLC26A8, 1:50) overnight at 4 °C. The slides were washed in 1× PBS, incubated with DyLight 488- or DyLight 594-labeled secondary antibodies (1:1000, Thermo Fisher) for 1 h at room temperature, and then counterstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma- Aldrich).
2.5 Electron Microscopy
For scanning electron microscopy (SEM), the sperm cells were fixed onto slides using 4% glutaraldehyde refrigerated overnight at 4°C. After washing the slides with PBS three times, the slides were gradually dehydrated with an ethanol gradient (30%, 50%, 75%, 95%, and 100% ethanol) and dried by a CO2 critical-point dryer. After metal spraying by an ionic sprayer meter, the samples were observed by SEM (S-3400, Hitachi). For transmission electron microscopy (TEM), the sperm cells were washed three times and fixed routinely. After embedded in Epon 812, ultrathin sections were stained with uranyl acetate and lead citrate, and observed under a TEM (TECNAI G2 F20, Philips) with an accelerating voltage of 80 kV.
RESULTS