Macrophage Recruitment and NLRP3 Inflammasome Activation

Immunofluorescence staining was conducted as described previously [9 (link)]. The slides were subjected to double immunofluorescence staining for NLRP3 (1:100, Abcam) and F4-80 (1:100, Abcam) in the aortic root to verify the recruitment of macrophages. NLRP3 (1:100, Adipogen) and Cleaved caspase-1 antibodies (1:100, ImmunoWay) were used to confirm the activation of the NLRP3 inflammasome. Cleaved caspase-1 (1:100, ImmunoWay) and TdT-mediated dUTP nick end labeling (TUNEL, Beyotime) were used to distinguish pyroptotic cells from apoptotic cells. Double staining with p22^phox or p67^phox (1:100, Santa Cruz Biotechnology) and CT-xB (1:100, Abcam, Cambridge, UK) was performed to verify the colocalization of NOX subunits with lipid rafts. Images were acquired using a laser scanning confocal microscope (LSM780, Zeiss, Jena, Germany).