Cy3 labelled secondary antibody

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.

IPTG (isopropyl-β-D-thiogalactopyranoside) and DTT were purchased from Sangon Biotech. Ni-nitrilotriacetic acid (Ni-NTA) was purchased from QIAGEN. Human plasminogen, ϵ-aminocaproic acid (ϵ-ACA), Creatinine Assay Kit, glass beads, urokinase-type plasminogen activator, anidulafungin, fluconazole, and DMEM medium were purchased from Sigma-Aldrich. Human Endothelial Serum Free Medium, HuMEC Basal Serum-Free Medium and Blood Urea Nitrogen Detection Kit were purchased from Thermo Fisher Scientific. Mouse nonspecific IgG2a was obtained from Invivogen. Cy3-labelled secondary antibody was purchased from Invitrogen. Chromogenic substrate D-Val-Leu-Lys-pNA·2HCl was purchased from Innovative Research. The LDH Cytotoxicity Assay Kit was obtained from Beyotime. PrimeScript ^TM RT Reagent Kit and the PrimeSTAR® Max DNA Polymerase were obtained from TaKaRa Bio. Rabbit anti-plasminogen antibody was obtained from Acris Antibodies. Anti-actin monoclonal antibody, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG were obtained from Abcam. HRP-labelled goat anti-mouse antibody was purchased from Dingguochangsheng Biotechnology. pET-21a (+) was purchased from Novagen.