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