The arteries were isolated, fixed in 4% paraformaldehyde and then embedded in paraffin. The embedded arteries were cut into 5 mm sections, stained with H&E and then examined by light microscopy. Huawei P20 mobile phone was used to record images.
Formaldehyde-fixed paraffin sections were incubated with primary antibodies including anti-NEK7 (1:100, BioSS), anti-NLRP3 (1:100, CST), anti-ASC (1:100, Santa Cruz) and anti-caspase-1 (1:100, CST) antibodies overnight at 4℃ and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at 37℃. Huawei P20 mobile phone was used to record images.
Additionally, the immunofluorescence technique was applied. After antigen retrieval, the slides were incubated with the primary antibodies overnight at 4°C. Next, an antimouse or antirabbit fluorescein isothicyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibody (1:100, Invitrogen) was added for 2 hours at 37°C. Then 4',6-diamidino-2-phenylindole (DAPI) was added at a dilution of 1:20 000 for 5 min, and the slides were cover-slipped with SlowFade Antifade (Invitrogen). The images were taken at 100 and 200 power magnification using an ix70 microscope and Magnafire V.1.1 software (both Olympus), and Image Pro-Plus software was used to generate colour composite images.
Formaldehyde-fixed paraffin sections were incubated with primary antibodies including anti-NEK7 (1:100, BioSS), anti-NLRP3 (1:100, CST), anti-ASC (1:100, Santa Cruz) and anti-caspase-1 (1:100, CST) antibodies overnight at 4℃ and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at 37℃. Huawei P20 mobile phone was used to record images.
Additionally, the immunofluorescence technique was applied. After antigen retrieval, the slides were incubated with the primary antibodies overnight at 4°C. Next, an antimouse or antirabbit fluorescein isothicyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibody (1:100, Invitrogen) was added for 2 hours at 37°C. Then 4',6-diamidino-2-phenylindole (DAPI) was added at a dilution of 1:20 000 for 5 min, and the slides were cover-slipped with SlowFade Antifade (Invitrogen). The images were taken at 100 and 200 power magnification using an ix70 microscope and Magnafire V.1.1 software (both Olympus), and Image Pro-Plus software was used to generate colour composite images.