Immunohistochemistry and double immunofluorescence staining

Immunohistochemistry was conducted as described previously [13, 20]. Mouse tissues were fixed in 60% methanol and 10% acetic acid in H₂O and paraffin embedded. Following deparaffinization and rehydration, tissue sections were treated with 3% hydrogen peroxide solution for 20 min to block endogenous peroxides activity. Antigen retrieval was carried out by treatment of the slides with 10 mM sodium citrate buffer (pH 6.0) or EDTA (pH 9.0) by microwaving for 10 min. The samples were blocked with 5% FBS in 0.1% PBS/BSA and incubated with primary antibody overnight at 4 °C. The standard streptavidin–biotin linked horseradish peroxidase technique was applied with 3,30-diaminobenzidine tetrahydrochloride for the development of peroxidase activity. The sections were counterstained with hematoxylin.

Double immunofluorescence staining was performed as described previously [13]. Primary antibodies used include the following: anti-α-smooth actin (Santa Cruz Biotechnology, sc-130616), anti-MCP1 (R&D systems, 479-JE-010; Novus Biologicals, NBP2-22115), anti-active caspase 1 (Invitrogen, AAC504N), anti-NLRP3 (AdipoGen, AG-20B-0014), anti-interleukin-1β (R&D systems, AF-401-NA), anti-LC3 (Sigma, L8918), anti-p62 (MBL, PM045), anti-Mac3 (BD Pharmingen, #550292), anti-ICAM1 (R&D systems, AF796) and anti-cleaved caspase 3 (Cell Signaling Technology, #9661). Images were captured through a Nikon upright microscope with an objective set to ×4 or ×20 magnification, quantified using CellSens Standard software, and expressed as percent of plaque area (%) = (staining positive area/plaque area) × 100%.