Er0815

The total protein of HHNs was extracted using a total protein extraction kit (#BB-3101; BestBio, Shanghai, China) and the concentration of isolated total protein was measured using a BCA protein quantification kit (#BB-3401; BestBio). Next, equivalent amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE, #YJ0014B; Yiji, Shanghai, China) and transferred onto a polyvinylidene difluoride (PVDF, #SD7966555; Yiji) membrane. Subsequently, the PVDF membrane was blocked with 5% skim milk at room temperature for 1 h, then treated with primary antibodies against NF-κB (1:1,000, #ER0815; HUABIO, Hangzhou, China), anti-NLRP3 (1:1,000, #ER1706-72; HUABIO), anti-caspase-1 (1:1,000, #ET1608-69; HUABIO), anti-IL-1β (1:1,000, #ET1701-39; HUABIO), anti-IL-18 (1:1,000, #EM170401; HUABIO), anti-TIGAR (1:1,000, #ER62495; HUABIO), and anti-GAPDH (1:1,000, #ER1901-65; HUABIO) overnight at 4°C. The next day, a secondary antibody (1:1,000, #HA1024; HUABIO) was used to treat the PVDF membrane at 37°C for 1 h. Protein bands were visualized using an ECL chemiluminescence detection kit (#BB-3501; BestBio). ImageJ was used to analyze the gray values of bands.

Paraffin sections (4 µm) were first dewaxed in xylene I, II, and III and then rehydrated in 100%, 95%, 90%, 80%, and 70% ethanol. Then, the samples were boiled in 1X sodium citrate, maintained at a subboiling temperature for 10 min to repair antigen and cooled to room temperature. Afterwards, endogenous peroxidase enzyme was inactivated using 3% H₂O₂ in methanol for 10 min in the dark at room temperature. Then, the sections were washed three times with PBS, sealed with 5% normal goat serum/1× PBS for 2 h at room temperature, and incubated with the primary antibody diluted in 2.5% normal goat serum/1× PBS overnight at 4°C. The primary antibody was a rabbit anti-NF-kB antibody (HuaBio, ER0815, 1:200 dilution). On the second day, the cells were washed three times with 1× PBS, incubated with a biotinylated goat anti-rabbit secondary antibody for 2 h at room temperature, washed three times with 1× PBS, and stained with DAB for 10 min. The reaction was stopped in ice water. Then, the samples were counterstained with hematoxylin, dehydrated, paraffinized and finally mounted and sealed with neutral gum. The dyed sections were photographed with an optical microscope (Nikon Eclipse Ni-U; Nikon, Tokyo, Japan).