Bca protein detection kit

Exosomes were fixed with 2% paraformaldehyde and placed on a Formvar-carbon-coated electron microscope grid. As a control, the grids were negatively stained and embedded with 1%(w/v) uranyl acetate, and incubated at 4°C for 10 min. The excess fluid was removed, the mesh was sucked dry by Whatman filter paper, and imaged in JEM-200CX electron microscope (JEOL Ltd., Tokyo, Japan). The Total protein contents of exosomes were measured with the BCA protein detection kit (Abcam). The size distribution of exosomes was tracked by Nanoparticle tracking analysis (NTA).

Kidney tissues were lysed with RIPA buffer to obtain total protein. After the protein concentration was determined by the BCA protein detection kit (Abcam, UK), the total protein was performed with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and electrotransfer. Then, the membrane is sealed with 5% completely skimmed milk powder, add the diluted primary antibody solution (NF-κB p65, 6535-1-Ig, Proteintech), (NLRP3, DF7438, Affinity), (caspase-1, AF5418, Affinity), (α-SMA, AF1032, Affinity), (smad3, AF6362, Affinity), (p-smad3, AP0727, Abclonal) and incubate the membrane at 4 °C for 12 h. Next, the membrane was washed with PBST (every 15 min) four times, then incubated with the diluted secondary antibody at room temperature for 2 h to enhance the chemiluminescence detection system immune signal. β-actin as a reference and Image J software was used for optical density analysis.