Ix83 dsu

After incubation in the appropriate medium, cells were fixed in 0.1% glutaraldehyde and 1.5% formaldehyde in PBS for 1 h and collected by centrifugation at 1300g for 5 min. The sperm pellet was immediately resuspended and incubated with 50 mM NH₄Cl in PBS during 15 min and washed twice by resuspension/centrifugation in PBS and once in distilled water. Water-resuspended cells were used to prepare smears on glass slides, which were air-dried at room temperature. Sperm were rinsed with PBS for 7 min and then permeabilized using acetone at −20 °C for 7 min and washed three times in PBS. Slides were then incubated with TRITC-phalloidin (1:30) in PBS for 1 h at room temperature in humid conditions, in the dark. Sperm were washed three times with PBS, once in distilled water and air-dried at room temperature. Finally, they were mounted under coverglass slides using Vectashield mounting media. Nonspecific staining was determined by incubating the sperm in the absence of TRITC-phalloidin. Slides were examined using a fluorescence confocal microscope (Olympus IX83-DSU), and images were captured at 60x magnification (Plan Apo, NA = 1.42 [oil]), with a sCMOS camera (Andor, Zyla). The fluorescence intensity was quantified using ImageJ software1.47 V (National Institute of Health) and was calculated in regions of interest localized in the sperm head. The background intensity was subtracted.