3 nit

Paraffin kidney sections from mice or humans (4 μm) were deparaffinized and rehydrated, and antigen retrieval was conduction utilizing 10 mM sodium citrate or EDTA for 15 min within a microwave oven. After blocking with 3% H₂O₂/PBS and 5% bovine serum albumin (BSA)/PBS, the following primary antibodies were incubated overnight at 4 °C: rabbit mouse anti-human/mouse LIGHT, LTβR, KIM-1, 3-NIT, 4-Hydro, Drp1, Mfn2, Bcl2, α-SMA (1:200, Abcam, Cambridge, UK), and rabbit anti-mouse/human HVEM (1:200, Santa Cruz, USA). Sections were then incubated with horseradish peroxidase-labeled goat anti-rabbit/mouse secondary antibodies (1:800, Beyotime, Shanghai, China) for 1 h at 25 °C. Following washing three times with PBS, 3,3’-diaminobenzidine chromogen solution (DAB, Beyotime, Shanghai, China) was used for visualization, and the sections were counterstained with hematoxylin and analyzed with light microscopy (Olympus, Tokyo, Japan). At least six viewing fields were randomly selected to quantify the number of positive areas (0.04 mm²).

Kidney cryosections (8 μm) were immersed into cold acetone for 15 min and then blocked with 5% BSA for 1 h at 25 °C. After incubated with primary antibodies against Ly-6G or F4/80 (1:100, Abcam, Cambridge, UK) overnight at 4 °C, sections were incubated with Dylight488-conjugated secondary antibody (1:200, Biolegend, San Diego, CA, USA), and then the nuclei were co-stained with Hoechst 33258 (5 μg/mL, Beyotime, Shanghai, China). The cryosections were tested using fluorescence microscopy (Olympus, Tokyo, Japan). For ICC experiments, the indicated coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.1% TritonX-100, and blocked with 5% BSA. The slips were incubated with primary antibodies against 3-NIT, 4-Hydro, Drp1, Mfn1, or MFF (1:100, Abcam, Cambridge, UK) overnight at 4 °C. In some experiments, mitochondria were labeled with Mito-Tracker Red CMXRos before fixation (Beyotime, Shanghai, China) according to the provided protocols. The next day, slips were stained with Cy3/ Dylight488-conjugated secondary antibody (1:200, Biolegend, San Diego, CA, USA), and then nuclei were co-stained with Hoechst 33258 (5 μg/mL, Beyotime, Shanghai, China). Quantification of positively stained cells was performed as described previously.