Indirect immunofluorescence
Indirect immunofluorescence (IF) is a common method for detecting
protein expression and localization in cells. In this study, the cell
suspension was first centrifuged at 2800 rpm, 4°C for 5 min, the
supernatant was discarded, and fixed with 2 ml of 4% paraformaldehyde
(Sigma-Aldrich) for 10 min at room temperature. Cells were then
incubated with 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 30 min. We
removed 0.5% TritonX-100, washed with PBS-T, added goat serum blocking
solution dropwise, and incubated in a 37°C incubator for 30min. After
blocking, we directly suck out the goat serum blocking solution without
washing. Then, enough primary antibody (p-IKBα, 1:300, bs-5515R, Bioss;
p-NF-κB, 1:100, BS4135, Bioworld) was added dropwise, covered, and
incubated in a 37°C incubator for 1 hour. The primary antibody was
removed, and enough immunofluorescent secondary antibody (goat
anti-rabbit IgG (FITC) 1:400) was dropped, for 30 minutes in the dark at
37°C. Finally, we mounted the slides with anti-fade mounting medium
(containing DAPI). Fluorescent sections were scanned using a digital
slide scanner (Pannoramic MIDI).