After treatment, BV-2 cells were fixed with 4% paraformaldehyde for 20 min then permeated by 0.3% Triton X-100 for 10 min and subsequently blocked by 1% BSA for 20 min. Primary antibodies (galectin-3, ab76245, Abcam, 1:400; lysosomal associated membrane protein 1, LAMP1, ab208943, Abcam, 1:400; Lipid A, GTX40001, GeneTex, 1:400; cytosolic phospholipase A2-α, cPLA2, sc-454, Santa Cruz, 1:100; Cyt c, 66264–1, Thermo Fisher, 1:400) were incubated with cells overnight at 4°C. Fluorescence conjugated secondary antibodies (Jackson ImmunoResearch, 711-025-152, 705-095-147, 115-095-003, 715-585-150, 1:200) were added at room temperature for one hour.
After fixing with 4% paraformaldehyde, cells were stained with BODIPY™ 493/503 (Invitrogen, Life Technologies, Carlsbad, CA, USA) or PI (Beyotime, Shanghai, China) for 20 min at room temperature. TUNEL staining (MK1027, BOSTER, Wuhan, China) was performed following the manufacturer’s instructions.
Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI), and all images were captured with fluorescent microscopy (Olympus BX41).
After fixing with 4% paraformaldehyde, cells were stained with BODIPY™ 493/503 (Invitrogen, Life Technologies, Carlsbad, CA, USA) or PI (Beyotime, Shanghai, China) for 20 min at room temperature. TUNEL staining (MK1027, BOSTER, Wuhan, China) was performed following the manufacturer’s instructions.
Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI), and all images were captured with fluorescent microscopy (Olympus BX41).