Fluorescence microscopy

Liver in situ O₂^·− production was determined by fluorescence probe DHE labeling. Frozen liver sections were prepared for immediate DHE staining. Thawed sections were incubated with 2 μM DHE at 37°C for 30 minutes (avoiding light). After washed 3 times by PBS, sections were immediately imaged using fluorescence microscopy (Olympus, Tokyo, Japan) and quantified by ImageJ software.
Intracellular O₂^·− levels were detected using the DHE staining. Cells were incubated with PBS diluted DHE (10 μM) at 37°C for 20 min (avoiding light), washed with PBS for 3 times, and then imaged using fluorescence microscopy (Olympus, Tokyo, Japan). Mitochondrial ROS in HepG2 cells was measured by MitoSOX Red at a concentration of 4 μM for 20 min at 37°C, imaged using fluorescence microscopy (Olympus, Tokyo, Japan), and quantified by ImageJ software.
DCFH-DA fluorescent probe was used to detect intracellular ROS generation. Cells were washed with PBS after incubation with DCFH-DA (10 μM) at 37°C for 30 min (avoiding light). Fluorescence intensity was measured at 530 nm with an excitation wavelength of 485 nm using a fluorescence microscope (Tecan, Crailsheim, Germany).

The lumbosacral spinal cords were extracted and fixed as above, before dehydrating in 15% sucrose solution for 4 h, 20% sucrose solution for 10 h, and 30% sucrose solution overnight at 4 °C. Finally, the spinal cords were embedded in OCT and stored at −80 °C. For immunofluorescence assay, 5-μm thick sections from all of the groups were prepared, washed with TBST for 15 min, fixed with paraformaldehyde for 10 min, permeabilized with 0.3% Triton X-100 for 15 min, blocked with 10% normal donkey serum in TBST for 1.5 h, and incubated overnight at 4 °C with primary antibodies against NLRP3 (rat IgG, 1:150), ASC (rabbit IgG, 1:300), caspase-1 (mouse IgG, 1:80), cleaved IL-1β (rabbit IgG, 1:100), GSDMD (rabbit IgG, 1:400), and Iba-1 (goat IgG, 1:300). Following incubation, the sections were incubated with Alexa Fluor 488-, 594-, or 647-conjugated secondary antibody (1:400), before DAPI was used to stain the nuclei. Spinal cord tissues were visualized and captured with a confocal laser microscope (Nikon A1, Nikon, Tokyo, Japan) or fluorescence microscopy (Olympus, Tokyo, Japan) at the same parameters. Immunofluorescence images of cells were captured via fluorescence microscopy (Olympus, Tokyo, Japan). Immunofluorescence was analyzed by ImageJ (National Institutes of Health, Bethesda, MD) on six lumbar spinal cord sections for each littermate.