P0123

After being treated as described in method 2.6, cells were washed using 0.5% PBST and fixed with paraformaldehyde of 4% for 10 min, followed by being washed using 0.5% PBST three times and blocked using immunol staining blocking buffer (P0102, Beyotime Institute of Biotechnology, Shanghai, China) for 1.2 h. Then the cells were incubated with Nrf2 rabbit anti-human primary antibody (1:100 dilution) at 4°C overnight, followed by being kept at room temperature for 1 h and washed using 0.5% PBST three times. The cells were subsequently incubated with FITC labeled goat anti-rabbit secondary antibody (1:200 dilution) at 37°C in a humidified incubator for 1.5 h, followed by being washed using 0.5% PBST three times. Finally, cells were stained with Hoechst 33258 of 20 μM for 5 min, followed by being washed using 0.5% PBST three times and adding antifade polyvinylpyrrolidone mounting medium (P0123, Beyotime Institute of Biotechnology, Shanghai, China). Then the slices were observed via a fluorescence microscope (Leica DMI400B).

For the acrosome staining, a protocol described by Zeng et al (2001) [26 (link)] was used after a slight modification. Briefly, 30 μL of the sperm samples were smeared onto a clean glass slide, air-dried, then fixed with absolute methanol for 10 min. Then, additional 30 μL of fluorescein isothiocyanate-peanut agglutinin (FITC-PNA, Sigma) solution (100 μg/mL) dilutes in PBS were spread over each slide. The slides were incubated in a dark and moist chamber at 37°C for 30 min, and subsequently washed with PBS twice and air-dried in the dark. Ten microliters of antifade solution (P0123; Beyotime, China) was added to the slide to preserve fluorescence before a clean coverslip was applied. The edges of the coverslip were sealed with colorless nail polish.
The acrosomal status of the spermatozoa was monitored and photographed by an epifluoresence microscope (Nikon 80i; Tokyo, Japan) with a set of filters (400X) with 488 nm excitation and 525 nm emission. For the same field, photographs were also taken with a phase-contrast microscope. At least 200 spermatozoa per slide were counted. Three separate aliquots (replicates) were assessed from each semen sample. All samples were barcoded and evaluated by one observer.