Nf κb p65

Occludin and NF-κB P65 (Bioswamp, Wuhan, China) were used as primary antibodies for immunohistochemical staining. Sections were incubated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity and washed with PBS three times. Then, a 10% goat serum blocking solution was added for 10 min to block excessive protein. The sections were incubated with primary antibodies (1 : 100) overnight. After washing three times with PBS, the sections were incubated with conjugated HRP (1 : 1000) for 10 min. Then, coloration was conducted using a 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) chromogenic reagent.

Proteins were extracted from the hippocampal tissue and BV2 cells. Lysis buffer and kits were used for quantification (Solarbio, Beijing, China). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) (Millipore, US). Blocking was performed with 5% skimmed milk, after which the PVDF was incubated for 12 h with primary antibodies against Bax (Bioswamp, Wuhan, China), Bcl-2 (Bioswamp), CREB (Bioswamp), p-CREB (Bioswamp), BDNF (Bioswamp), ERK1/2 (Bioswamp), p-ERK1/2 (CST), p38MAPK (Bioswamp), p-p38MAPK (Abcam), TLR4 (Bioswamp), NF-κB p65 (Bioswamp), p-NF-κB p65 (Abcam), and GAPDH (Bioswamp). The membranes were incubated with a secondary antibody (Bioswamp) for 2 h, and the protein bands were scanned.