Fluorescent rabbit secondary antibody

Cells were treated according to grouping requirements, washed three times using PBS, fixed with 4% paraformaldehyde (MACKLIN, Shanghai, China) for 20 min at room temperature, and permeabilized with 0.5% immunostaining permeabilization buffer with Triton X-100 (Beyotime, Shanghai, China) for 20 min. Thereafter, cells were blocked with 5% BSA for 30 min and incubated overnight at 4 °C with rabbit anti-P65 antibody (CST, Shanghai, China). Cells were then washed three times with PBS and labeled with the corresponding fluorescent rabbit secondary antibody (Invitrogen™, Shanghai, China) at a dilution of 1:800 for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; CST, Shanghai, China) for 10 min. Final images were taken using a multifunctional microplate detection system (CYTATION5, BIOTEK, USA).

The paraffin sections were deparaffinized and rehydrated with gradient alcohol. After rinsing with 0.01 mol/L PBS for 3 times, the slides were blocked by 10 % serum in a humid box at 37 ^oC for 30 min. The primary antibody (Table S3) was added and then incubated in a 37 ^oC humid box for 1 h and washed with PBS. Then fluorescent rabbit secondary antibody (1:200, Invitrogen) was dropped onto the slide that was subsequently incubated in a humid box in the darkness for 1 h. DAPI was added dropwise and the sections were incubated for 5 min in the dark. Finally, the slides were mounted with glycerol and the resulted slides were immediately observed and photographed by a fluorescence microscope (CKX41, Olympus, Japan).