Immunofluorescence was performed to detect the level of NLRP3 in kidney tissues. After antigen retrieval, the sections were blocked with 1% bovine serum albumin for 15 min, followed by incubation with primary antibody against NLRP3 (1:200; Affinity Biosciences) at 4°C overnight. Then, the sections were incubated with Cy3-labeled secondary antibodies (1:200; Invitrogen, ThermoFisher Co., Ltd) for 60 min. After counterstaining with DAPI, the sections were observed under a fluorescence microscope (Olympus) and images were captured at 400× magnification.
For fibronectin detection in HK-2 cells through immunofluorescence, HK-2 cells were seeded onto glass slides and then subjected to indicated treatments. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. After blockade with 1% bovine serum albumin, the cells were incubated with primary antibody against fibronectin (1:200, Affinity Biosciences) at 4°C overnight. Thereafter, Cy3-labelled secondary antibody (1:200, Invitrogen) was added onto cells and incubated for 60 min at room temperature. Cell nuclei were stained with DAPI (Aladdin Biochemical Technology Co., Ltd). The cells were observed under a fluorescence microscope and images were captured at 400× magnification.
For fibronectin detection in HK-2 cells through immunofluorescence, HK-2 cells were seeded onto glass slides and then subjected to indicated treatments. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. After blockade with 1% bovine serum albumin, the cells were incubated with primary antibody against fibronectin (1:200, Affinity Biosciences) at 4°C overnight. Thereafter, Cy3-labelled secondary antibody (1:200, Invitrogen) was added onto cells and incubated for 60 min at room temperature. Cell nuclei were stained with DAPI (Aladdin Biochemical Technology Co., Ltd). The cells were observed under a fluorescence microscope and images were captured at 400× magnification.