Nis element ar 4

10 μm Frozen sections of testis obtained with cryostat microtome and Sertoli cells were fixed in 4% PFA for 15 min and permeabilized in 0.1% Triton X-100 in PBS for 10 min. Then sections and cells blocked in 0.5% BSA in PBS for 1 h at room temperature, followed by an overnight incubation of primary antibodies at 4 °C. The primary antibodies used were LC3 (1:100, ab128025, Abcam), p62 (1:200, ab91526, Abcam), SOD (1:200 ab16831; abcam), Anti-Heme Oxygenase 1 (1:200; ab68477; abcam), β-catenin (1:100, ab32572, Abcam), Cx43 (1:100, ab11370, Abcam), and ZO-1 (1:100, 61–7300, Invitrogen). Following a PBS wash, the sections were incubated with goat anti-rabbit (CW0159S) or mouse (CW0145S), Cy3 Conjugated (CwBio, China) for 1 h at room temperature. The nuclei were stained with hoechst 33342 (C1022, Beyotime, China) for 1 h. Images were obtained with an A1R Confocal microscopy system (Nikon, Tokyo, Japan) and prepared with Nikon NIS-element AR 4.0 software.

C3H10T1/2 cells (1×10⁵ cells/well) were plated in 24-well plates on 1×1 cm² glass coverslips. Then, the cells were fixed in absolute acetone for 15 min at 4°C. Following 3 washes with PBS, the cells on the glass coverslips were blocked with goat serum (dilution, 1:20, ZSGB-BIO, Beijing, China), washed again, and incubated with the primary anti-cardiac troponin T(cTnT) monclonal antibody (ab209813; 1:400; Abcam, Cambridge, MA, USA) overnight at 4°C. Then, the cells were washed with PBS and incubated with a Cy3-conjugated secondary antibody (CW0159S; 1:150; Beijing Cowin Bioscience Co., Ltd., Beijing, China) for 1 h at 37°C. Following washing with PBS, 4′,6-diamidino-2-phenylindole was added for 3 min. Following the final wash, images were acquired under a fluorescence microscope (BX51; Olympus Corporation, Tokyo, Japan) and prepared by Nikon NIS-element AR 4.0 software. A total of six fields of view were assessed, and three replicates were performed.