Kidneys samples were fixed in 10% neutral formalin and paraffin-embedded sections were stained Periodic acid-Schiff (PAS) and Sirius red. Samples were scored by an experienced pathologist in a blinded fashion for signs of acute tubular injury. Fibrosis was quantitated in Sirius red-stained sections using MRI fibrosis tool and Color Deconvolution plugin for “ImageJ [https://github.com/MontpellierRessourcesImagerie/imagej_macros_and_scripts/wiki/MRI_Fibrosis_Tool ]”. Ten similarly oriented photos in the same part of the kidney at the same magnification were studied per biological sample. Color deconvolution and simple auto-thresholding were applied and the fibrosed area of the selection was measured and compared to the area of the selection on the input image.
Immunofluorescence staining was performed after deparaffinization and antigen retrieval using citrate buffer pH 6.0 with a pressure cooker (PickCell Laboratories, Agoura Hills, CA). Antibodies were diluted in blocking buffer (TBS 0.1% Tween 20, 0.05% Triton X-100, 5% bovine serum albumin) as follows: anti-GSDMD (1:100, Santa Cruz, #393656), anti-SLC34A1 (1:100, Novus, #NBP2-13328), anti-CD11B (1:50, BD, #555386), AF555-conjugated donkey anti-rabbit (1:1000, LifeSciences, #A31572), AF488-conjugated goat anti-mouse (1:1000, LifeSciences, #A11029).
Immunofluorescence staining was performed after deparaffinization and antigen retrieval using citrate buffer pH 6.0 with a pressure cooker (PickCell Laboratories, Agoura Hills, CA). Antibodies were diluted in blocking buffer (TBS 0.1% Tween 20, 0.05% Triton X-100, 5% bovine serum albumin) as follows: anti-GSDMD (1:100, Santa Cruz, #393656), anti-SLC34A1 (1:100, Novus, #NBP2-13328), anti-CD11B (1:50, BD, #555386), AF555-conjugated donkey anti-rabbit (1:1000, LifeSciences, #A31572), AF488-conjugated goat anti-mouse (1:1000, LifeSciences, #A11029).