Nuclear Translocation of NF-κB p65 in hBMECs by Immunofluorescence Analysis

The cells were seeded at a density of 1.0 × 10⁵ on glass coverslips and treated with medium (vehicle control), AS (25, 50, and 100 μM), or TAK-242 (1 μM) for 12 h. Then, the cells were washed with PBS for 15 min at room temperature after 12 h treatment and treated with Aβ_1-42 (50 μM) or same volume of medium for another 24 h. The cells were fixed in 4% paraformaldehyde for 30 min at 4°C and washed with PBS for three times with 5 min for each time, then the cells were incubated with 0.1% Triton X-100 for 10 min, and blocked with 1% bovine serum albumin (BSA) for 30 min. The cells were incubated with rabbit monoclonal primary antibody (1:500) against NF-κB p65 at 4°C overnight, followed by HRP-conjugated goat anti-rabbit anti-NF-κBp65 secondary antibody (1:2000) for 2 h and washed with PBS for 15 min. The cells were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 10 min and washed with PBS for 15 min. The signals were detected and assessed under an inverted phase contrast fluorescence microscope (LD laser: 405 nm, 25 mW; multi-line Ar laser: 458, 488, and 515 nm, 40 mW; HeNe green laser: 543 nm, 1 mW; and HeNe red laser: 635 nm, 20 mW) at ×400 magnifications (OLYMPUS company, model: CKX41, Shinjuku-ku, Tokyo, Japan). All experiments were performed three times in triplicate.