Ct xb

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).

Immunofluorescence staining method was used according to our previous study (Wang R. et al., 2017 (link)). Specific primary antibodies were double stained with frozen aortic root sections for macrophage accumulation analysis, including goat anti-NLRP3 (Abcam) and rat anti-F4-80 (macrophage marker; Abcam) antibodies. goat anti-NLRP3 (Abcam) and rabbit anti-cleaved caspase-1 antibodies (ImmunoWay, TX, United States) were used for NLRP3 inflammasome activation analysis. Antibodies were double stained with α1-nAchR and CT-XB (lipid raft marker; Abcam) for correlation analysis. The analysis was imaged under a laser scanning confocal microscope (Zeiss, Germany).