Mice were anesthetized and perfused with PBS. The kidneys were collected, fixed with 4% PFA, and cut to obtain 30 μm sections using a vibratome (Invitrogen, U.S.A). After three rinses in PBS (5 min each), the sections were blocked in PBST (PBS + 0.3% Triton X-100) with 3% BSA for 2 h, and were then incubated in primary antibodies rabbit anti-lamin B1 (Abclonal, China), mouse anti-p53 (Abcam, UK) or rabbit anti-p21 (ABclonal, China) with a dilution of 1:1000 at 4 °C for 24 h, followed by three rinses in PBS. Then, the sections were incubated with goat anti-rabbit IgG H&L Alexa Fluor® 488 (1:300, Abcam, UK) or goat anti-mouse IgG H&L Alexa Fluor® 488 secondary antibodies (1:300, Abcam, UK) for 2 h at room temperature. After the incubation, the sections were washed with PBS for 5 min, and the nuclei were counterstained with DAPI for 5 min. Finally, the sections were washed in PBST and mounted in mounting medium. Fluorescence images were acquired with a 20 × objective on a confocal microscope.
Flies were anesthetized with CO2, dissected in PBS, and fixed with 4% PFA at room temperature. Brain sections were prepared and incubated with rabbit anti-caspase-3 primary antibody (1:500, Beyotime, China) and then with goat anti-rabbit IgG H&L Alexa Fluor® 488 (1:200, Abcam, UK), followed by DAPI staining and fluorescence imaging.
Flies were anesthetized with CO2, dissected in PBS, and fixed with 4% PFA at room temperature. Brain sections were prepared and incubated with rabbit anti-caspase-3 primary antibody (1:500, Beyotime, China) and then with goat anti-rabbit IgG H&L Alexa Fluor® 488 (1:200, Abcam, UK), followed by DAPI staining and fluorescence imaging.