Nlpr3

4 μm thick kidney sections embedded in paraffin were prepared for immunohistochemistry assays. Sections were rehydrated, and antigens retrieved using heated citrate. Primary antibodies including NLPR3 (Adipogen, USA) and IL-1β (Miltenyi, China) were applied in blocking solution overnight at 4°C. After being incubated with anti-rabbit IgG (1 : 100; Beyotime) for 30 min at 37°C and washed and developed with DAB (Bioss, China), the stained sections were examined using a light microscope (Nikon Eclipse TE2000-U, NIKON, Japan) at 400x magnification. The semiquantitative immunohistochemical analysis was scored using Image-Pro plus 6.0 software in ten randomly selected cortical sections in each tissue section.

Paraffin-embedded sections from the kidney cortex were used for immunohistochemistry. Briefly, the sections were incubated with primary antibodies to F4/80, neutrophil (Abcam, USA), NLPR3 (Adipogen, USA), ASC, caspase-1 (Santa Cruz Biotechnology, USA) overnight at 4 °C. The sections were then analyzed using streptavidin peroxidase detection system (Maixin, China) according to the manufacturer’s protocol. The reaction was developed using DAB substrate kit (Maixin, China), and counterstaining was performed using hematoxylin.
For analysis and localizing the expression of the NLRP3 and cathepsin B, immunofluorescence staining of tissue sections or formaldehyde-fixed cells was performed using anti-NLRP3, anti-cathepsin B antibodies (Abcam, USA), respectively in a humidified chamber overnight at 4 °C, followed by incubation with an Alexa fluorescein-labeled secondary antibodies (Invitrogen, USA) for 1 h. Cell nuclei were stained with DAPI. Immunostained samples were visualized under a confocal microscope. Immunofluorescence for p65 (Cell Signaling Technology, USA) was similarly performed. Quantification of intensity of p65 in nuclear was performed by measuring area, integrated density, and mean gray value using Image-Pro Plus.