Phosphorylated nf κb p65 antibodies

After the slices were deparaffinized and rehydrated, the antigen was retrieved by citrate buffer. 3% H₂O₂ was employed to block endogenous peroxidase for 10 minutes, followed by blocking of the nonspecific protein docking site with 0.5% BSA for another 1 hour. After that, we inoculated the slices overnight with 1 : 200 diluted phosphorylated NF-κB p65 antibodies (Cell signaling Tech, United States) and Nrf2 antibodies (Cell signaling Tech, United States), respectively, at 4°C. After rinsing thrice with PBS, the matching secondary antibody was introduced and incubated for 1 hour. Images were observed under a microscope after nuclear counterstaining.

Total proteins from the lung tissue homogenates were extracted according to the instructions provided in the BCA protein assay kit (Beyotime, China). Fractionation of 40 μg of protein samples was done with the SDS-polyacrylamide gel and transfer-embedded onto PVDF membranes. Afterwards, blocking of membranes was done with the 5% skimmed milk for one hour and inoculated overnight with primary antibodies against NF-κB p65 antibodies (1 : 1000, Cell signaling Technology, United States), phosphorylated NF-κB p65 antibodies (1 : 1000, Cell signaling Technology, United States), Nrf2 antibodies (1 : 1000, Cell signaling Technology, United States), and Histone H3 (1 : 1000, Cell signaling Technology, United States) in TBST (Tris-buffered saline/Tween) at 4°C. Then, we rinsed the membranes thrice in TBST and inoculated for two hours with secondary antibodies at room temperature. Immobilon Western Chemiluminescent HRP Substrate (Millipore, United States) was adopted to detect positive immune reaction.