Anti ngal antibody

Mice kidney samples were incubated with H₂O₂ before treatment with anti-NGAL antibody (1:100, Abcam, USA), anti-MPO antibody (1:500, Servicebio, China) anti-Ly6G antibody (1:500, Servicebio, China) and anti-F4/80 antibody (1:500, Servicebio, China). The slices were washed by PBS before incubating with a secondary antibody. The positive areas of NGAL and positive cells of MPO, Ly6G and F4/80 were measured under a light microscope and calculated by ImageJ software (version 1.51, USA).

To determine the protein level of NGAL in kidney tissues or HK2 cells, renal cortical tissues or cells were homogenized using ice-cold RIPA lysis buffer (Applygen Biotechnology, Beijing, China) and quantified by the bicinchoninic acid assay (Applygen Biotechnology) with BSA as standard. Equal amounts of protein were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membrane. After blocking with 5% (w/v) skim milk solution for 1 h, the membranes were incubated with anti-NGAL antibody 1:1000 (Abcam plc, Cambridge, MA, USA) overnight. β-Actin (Santa Cruz Biotechnology, San Diego, CA, USA) was used as the internal standard. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody immunoglobulin G (IgG) at 1:2000 dilution for 1 h at 24–28 °C after being washed thrice using Tris-buffered saline with 0.1% Tween 20 (TBS/T). The immunoreactive bands were visualized using an enhanced chemiluminescence system (LAS 4000; GE healthcare, CA, USA). The intensity of the detected bands was analyzed using the Image J program (Version 1.38).