Nlrp3

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).

Liver tissues were embedded in paraffin, and slides were cut from the paraffin tissue. Slides were heated with sodium citrate for antigen retrieval and then incubated with primary antibodies including TLR2 (#GB11554, 1:300, Servicebio, Wuhan, China), NLRP3 (#YT5382, 1:100, ImmunoWay Biotechnology), ASC (#YT0365, 1:200, ImmunoWay Biotechnology), Caspase-1 (#GB11383, 1:500, Servicebio), and IL-1β (#GB11113, 1:250, Servicebio) overnight at 4°C. Secondary antibody HRP-conjugated Goat Anti-Rabbit IgG(H+L) (#GB23303, 1:200, Servicebio) was incubated for 50 min at room temperature. After PBS washing, peroxidase substrate DAB (#K5007, DaKo, Copenhagen, Denmark) was used as chromogen. The slices were stained, differentiated, and sealed before visualizing and taking photos under the microscope. Images of IHC were quantified using Image-Pro Plus 6.0 and assessed based on the sum value of integrated optical density (IOD, Area × Intensity) (32 (link), 33 (link)). The average numbers of IOD were counted from three randomly chosen fields.