L7381

Spermatozoa were collected from the caput, corpus, and cauda epididymis of adult mice. Immunofluorescence was enhanced as previously described [36 (link)]. Briefly, sperm cells on slides were fixed with 4% PFA, incubated with 0.5% Triton X-100, and blocked with 10% goat serum (AR1009, Boster Biological Technology Co., Ltd., Wuhan, China). The slides were then incubated with antibody against ATP1B3 (1:100; ab231671, Abcam, Cambridge, MA, USA) at 4 °C overnight, and anti-rabbit Alexa Fluor 568 (1:1000; ab175471, Abcam, Cambridge, MA, USA) was used as the secondary antibody for incubation at 37 °C for 1 h. Peanut agglutinin (PNA) is a lectin that binds specifically to the outer acrosome membrane of sperm [37 (link)]. Here, FITC-labeled PNA (PNA-FITC; 1:2000, L7381, Sigma-Aldrich, St. Louis, MO, USA) was used to stain the sperm acrosome and co-incubated with secondary antibodies. Finally, sperm nuclei were revealed using 4′,6-diamidino-2-phenylindole (DAPI; 1:1000, D9542, Sigma-Aldrich, St. Louis, MO, USA) stain at 37 °C for 10 min. Images were acquired with confocal laser scanning microscopy (Leica, Heidelberg, Germany).

The eyes were enucleated, fixed with 4% PFA for 30 min at room temperature, and immersed in OCT compound. Then, they were frozen and cut into 8-μm sections using a cryostat (CM1800, Leica Microsystems, Wetzlar, Germany) and mounted onto MAS-coated glass slides (MAS-01, Matsunami, Osaka, Japan). After removing the OCT compound in PBS containing 0.3% Triton X-100 for 15 min, the sections were blocked with 10% NGS in PBS for 1 h. Then, they were incubated with primary antibodies (chicken anti-vimentin [ab24525, Abcam], mouse anti-Rho [1D4, Abcam], FITC-conjugated peanut agglutinin (PNA) [L7381, Sigma-Aldrich], rabbit anti-PSD95 [monoclonal, 1/100, D27E11, Cell Signaling Technology, Danvers, MA, USA], rabbit anti-GS [ab73593, Abcam]) diluted in PBS at 4°C overnight. After washing with PBS, the sections were incubated with Alexa-conjugated secondary antibody (Goat anti-Rabbit IgG [H+L], Alexa Fluor™ 488, A-11008, Goat anti-Rabbit IgG [H+L], Alexa Fluor™ 546, A-11035, Goat anti-Mouse IgG [H+L], Alexa Fluor™ 647, A-21235) diluted in PBS for 1 h at room temperature, and the nuclei were counterstained with DAPI. Images were acquired using a BZ-X710 confocal microscope.

Immunostaining was performed according to standard protocols. Embryos were fixed in 4% PFA, paraffin embedded and sectioned. Sections were incubated in primary antibody overnight at 4°C. Secondary antibodies with fluorescent tags were then applied at 1:1000 along with Hoechst 33342 (1:2,000; Invitrogen) and incubated at room temperature for 1 hour. Slides were then washed and mounted with mounting media (ProLong Gold, Invitrogen). Antibodies used in this study included: mouse anti-PHH3 (1:500; 05–1336 Millipore), rabbit anti-CC3 (1:500; AF385 R&D Systems) and Peanut agglutinin, FITC conjugate (20 μg/ml; L7381 Sigma).

At least 20 embryos were fixed in 4% (W/V) paraformaldehyde, permeabilized with 1% Triton X-100 in PBS, blocked with 3% BSA for 1 h at room temperature (RT), respectively. For spindle shape detection, oocytes were stained with anti-α-tubulin-FITC antibodies (diluted 1:800; F2168, Sigma Aldrich) for 30 min. For CGs distribution detection, embryos were stained with anti-PNA-FITC antibodies (diluted 1:120; L7381, Sigma Aldrich) for 30 min. For autophagosome detection, oocytes were incubated with anti-LC3 antibodies (diluted 1:100; catalog no. 3868, Cell Signal Technology) and TOM 20 antibodies (diluted 1:100; catalog no. 12741, Cell Signal Technology) for 1 h. For FoxO3a detection, oocytes were incubated with anti-FoxO3a antibodies (diluted 1:100; catalog no. 12829, Cell Signal Technology) and TOM 20 antibodies (diluted 1:100; catalog no. 12741, Cell Signal Technology) for 1 h at RT and incubated with anti-rabbit IgG conjugated with Alexa Fluor 549 (red) and anti-mouse IgG conjugated with Alexa Fluor 488 (green) for another 1 h in the dark. The coverslips were finally mounted on glass slides with 10 mg/ml DAPI (Beyotime Institute of Biotechnology, Haimen, China). Fluorescent images were taken using a laser-scanning confocal microscope (Carl Zeiss, Göttingen, Germany).

Mouse eyes were enucleated and fixed with 4% paraformaldehyde (PFA) for 1 h at 4°C. After removal of the cornea and lens, the retinas were dissected from the posterior eye cup. Each retina was blocked for 1 h with PBS containing 10% nonfat dried milk and 0.3% Triton-X 100 (9002–93–1; Wako; Osaka, Japan), and then incubated with rabbit anti–Iba-1 antibody (1:100,019–19741, Wako) and FITC-conjugated PNA (1:100, L7381; Sigma-Aldrich) at 4°C overnight. After being washed with PBS, the retinas were incubated with appropriate Alexa Fluor 488- or Alexa Fluor 647-conjugated secondary antibodies (Invitrogen) for 1 h at RT and then mounted on slides. Immunofluorescence images were acquired using a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). The numbers of PNA⁺ cone photoreceptor cells and Iba-1⁺ microglia/mφ cells were counted in 0.015625-mm² retinal areas in the superior, inferior, temporal, and nasal areas located 250 and 500 μm from the optic disc by using Image J ver. 1.52a software (U.S. National Institutes of Health; NIH), and each number was averaged at both the 250 and 500 μm distances. The names and conditions of the samples were masked from the observers.