Anti cd81 h 121

An immunofluorescence assay was performed on epididymal, freshly ejaculated, frozen-thawed, capacitated and acrosomereacted sperm. The pellets of cryo-conserved sperm were washed twice in PBS at 200 g for 10 min at room temperature. After washing, the sperm suspension was smeared on slides and fixed by cold acetone-methanol (1:1) wet fixation and dried. All treatments were applied in a humid chamber to prevent the drying out of the cell smears. Sperm were blocked with Super Block Blocking Buffer (Thermo Scientific) for 1 h at 37°C and treated with the primary antibody anti-CD81 (H121; Santa Cruz Biotechnology) (1:100) for 1 h at 37°C in a humidified chamber. As a secondary antibody, goat anti-rabbit IgG-FITC was conjugated (1:200) (Vector Laboratories, Burlingame, CA, USA) for 30 min in the dark at room temperature. Nuclear DNA of both CD81-reactive and non-reactive sperm was stained by Vectashield mounting medium with DAPI (Vector Laboratories). The intactness of sperm acrosomes was assessed by Peanut agglutinin-TRITC (PNA-TRITC, Vector Laboratories). Immunostaining was evaluated under a Leica DM5500 B epifluorescence microscope at 400 and 1000× magnifications and the fluorescence images were recorded using a Leica DFC340 FX digital camera and processed using Leica Advanced Fluorescence software. Representative results are shown.