Ab31624

Sections were prepared from the rehydrated paraffin-embedded tissues using IHC, and the sections were incubated with antibodies against Ki67 (1:200, ab15580, Abcam, UK), p-p65 antibody (1:200, ab31624, Abcam, UK), and CK2α (1:200, PA5-28686, Thermo Scientific, Waltham, MA, USA). Sections were then stained with biotinylated anti-mouse and anti-rabbit secondary antibodies to IgG (1:200; Sigma-Aldrich) for 2 h and incubated with HRP-conjugated streptavidin for 1 h. Images were taken using a Leica DMI4000B microscope (Olympus Soft Imaging Solutions, Hamburg, Germany).

After lysing astrocytes in radioimmunoprecipitation assay lysis buffer (P0013C, Beyotime, China), the lysate was centrifuged at 12000×g for 30 min at 4°C and the supernatant was collected. After quantitatively balancing the histone concentrations, the sodium dodecyl sulfate (SDS) loading buffer was added, proteins were denatured at 95°C for 5 min, followed by electrophoresis on 10% SDS/PAGE gels (P0670, Beyotime, China), and then transferred to a nitrocellulose membrane. After blocking the nitrocellulose membrane (FFN08, Beyotime, China) with 5% bovine serum albumin (BSA, ST023, Beyotime, China) for 2 h, antibodies against P-IKKα/β (1:100, ab194528, Abcam, U.S.A.), IKKα (1:500, ab38515, Abcam, U.S.A.), IKKβ (1:500, ab124957, Abcam, U.S.A.), P-IκBα (1:100, ab133462, Abcam, U.S.A.), IκBα (1:500, ab32518, Abcam, U.S.A.), P-p65 (1:100, ab31624, Abcam, U.S.A.), p65 (1:500, ab32536, Abcam, U.S.A.), AQP4 (1:1000, ab259318, Abcam, U.S.A.), and tubulin (1:2000, ab7291, Abcam, U.S.A.) were added and incubated overnight at 4°C. The membrane was washed the next day, and horseradish peroxidase-labeled secondary antibodies (1:2000, ab288151, Abcam, U.S.A.) were added and incubated at room temperature for 2 h. After washing the membrane, the ECL developer was added, and the Gel Doc™ XR with an imaging system (Bio-Rad, U.S.A.) was used to record images.