Lr white resin medium grade

For post-embedding (on-section) immuno-gold EM the samples were fixed with the same primary aldehyde fixative but osmium fixation was omitted. The samples were stained en bloc with 2% aqueous uranyl acetate, dehydrated in 50% and 75% ethanol, and embedded in LR White resin medium grade (catalog # 14381-CA; Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were cut on Leica EM UC7 ultramicrotome and collected onto Formvar-carbon coated nickel grids. The grids were incubated in a wet chamber sequentially on drops of blocking buffer [0.1% bovine serum albumin (BSA) and 0.01 M glycine in 0.05 M TBS], then on rabbit anti-Vimentin (ab92547; Abcam) primary antibody at (1:500) dilution in 1% BSA in 0.05 M TBS (diluting buffer) for 1 h at room temperature and then overnight at 4°C. After washing in blocking buffer grids were incubated with secondary antibody [goat anti-rabbit IgG (H + L)] conjugated to 15 nm colloidal gold particles (catalog # 25113, code815.011; Electron Microscopy Sciences), which was diluted 1:20 in diluting buffer, for 1 h at room temperature. After washing in TBS and distilled water grids were fixed in 2% aqueous glutaraldehyde, washed, stained with uranyl acetate and lead citrate, and examined in the transmission electron microscope.

Mouse cauda spermatozoa prepared by the swim-out method were fixed in 4% paraformaldehyde for 30 min at room temperature. The spermatozoa were dehydrated in graded ethanol and centrifuged at 500 g. The pellet was then embedded overnight in LR white resin (medium grade; Electron Microscopy Sciences, Fort Washington, PA) at 56 °C. Ultrathin sections were prepared and mounted onto nickel grids. The sections were incubated with anti-mBPI-N serum (1:25) diluted in (PBS-T + 5% goat serum); otherwise, control sections were incubated with preimmune rabbit serum, followed by incubation with 10 nm gold-conjugated goat anti-rabbit IgG (GE Healthcare Bioscience, Carlsbad, CA, USA). Between each step, the grids were washed three times with PBS-T for 10 min at room temperature. After staining with uranyl acetate and lead citrate, the ultrathin sections were examined by H-600 electron microscope (Hitachi, Tokyo, Japan).