Rabbit anti nlrp3 polyclonal antibody

For the double staining of an indirect immunofluorescence assay of P2X7R/NLRP3 and podocyte marker synaptopodin, frozen renal tissues of rats were cut into 5 μm thick sections. A rabbit anti-P2X7R polyclonal antibody (1 : 100 dilution, Alomone) or rabbit anti-NLRP3 polyclonal antibody (1 : 100 dilution, Novus) and mouse anti-synaptopodin monoclonal antibody (1 : 50 dilution, Santa Cruz) were used as primary antibodies. Rhodamine-labeled goat anti-rabbit immunoglobulins (Beijing Zhongshan) and FITC-labeled goat anti-mouse immunoglobulins (Beijing Zhongshan) were used as secondary antibodies, respectively. After staining, the tissue sections were observed with a fluorescent microscope (Nikon, Japan).

Tissue and cell proteins were lysed on ice with radioimmunoprecipitation assay lysis buffer (Cwbio, Taizhou, China). Total proteins were collected after centrifugation. Then, 5 µL of 5× loading buffer was added to the proteins, which were denatured at 98°C and stored in a refrigerator at −20°C. Colloidal preparation, electrophoresis, membrane transfer, sealing, and other operations were carried out successively according to the kit’s instructions. The following primary antibodies were used during incubation: goat anti-IL-1β polyclonal antibody (1:1000, R & D Systems, USA), rabbit anti-CaSR polyclonal antibody (6D4, 1:500, Santa Cruz Biotechnology, USA), rabbit anti-NLRP3 polyclonal antibody (1:500, Novus, USA), mouse anti-caspase-1 monoclonal antibody (1:500, Novus, USA), and mouse anti-β-actin polyclonal antibody (1:500, Boster, China). Protein bands were detected using ECL (Pierce, USA).