Immunofluorescence microscopy

HT22 cells were seeded onto coverslips from six-well plates treated in accordance with the experimental design. At designated time points, the cells were treated as follows: fixed with 4% paraformaldehyde for 12 min, permeabilized for 10 min using 0.2% Triton X-100, and covered with goat serum (SL038, Solarbio). Then, the cells were incubated with LC3 (1:200), P62 (1:50), and LAMP-2 (1:200) overnight at 4° C, followed by incubation with secondary antibodies for 1 h, and labeling with DAPI (BD5010, Bioworld) for 40 min. Finally, the images were observed with a DMi8 fluorescence microscope and Leica X software at 800× magnification (DMi8, Leica, Germany).