Anti fading mounting medium

MRC-5 cells were plated in 48-well plates at a density of 1 × 10⁴ cells per well. After 48 h of cell treatment, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then rinsed with PBS. Subsequently, the cells were treated with 0.1% Triton-X for 20 min and rinsed again. Afterward, they were blocked with 5% bovine serum albumin (BSA, Solarbio, Beijing, China) for 60 min at room temperature, followed by incubation with antibodies (diluted 1:200) such as LC3-II, P62, etc., overnight at 4 °C. The cells were then rinsed and incubated for 1 h in the dark with secondary antibodies (goat anti-mouse antibody, Proteintech or goat anti-rabbit antibody, Proteintech). Subsequently, DAPI (Beyotime Biotechnology, Shanghai, China) was added to the cells for a 10-min incubation in the dark. Finally, the samples were sealed with an anti-fading mounting medium (Beyotime Biotechnology, Shanghai, China) and observed using an inverted fluorescence microscope.

Fixed low-passage poorly-differentiated human MEC cells from 2D and 3D culture were incubated overnight at 4 °C with mouse monoclonal antibodies against HIF1α (1:200; Abcam). After rinsing in PBS, cells were further incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:500; Abcam) and rinsed. Negative controls included omission of primary or secondary antibodies and substitution of primary antibodies with isotype controls. After mounting in anti-fading mounting medium (Beyotime, Beijing, China), stained cells were visualized and imaged with a fully motorized, Olympus IX83 inverted wide-field epifluorescence microscope with a DP80 CCD camera under the monochromatic mode controlled by Olympus cellSens Dimension software (Olympus; Tokyo, Japan).