Visualizing P65 Nucleation in RAW264.7 Cells

RAW267.4 cells were incubated on 24 mm × 24 mm coverslips in six-well plates at a density of 1 × 10⁵/well to observe their morphology and P65 nucleation. The culture conditions were the same as those applied to the BLANK, LPS, and LPS + KN-17 treatments for RAW264.7 macrophages. On days 1 and 3, the cells were placed in 4% (v/v) paraformaldehyde (PFA) and their membranes were ruptured with 0.25% (w/v) Triton X-100 for 3–5 min. Each cell group was then blocked with 1% (v/v) bovine serum albumin (BSA) for 30 min. P65 was stained with anti-P65 antibody (Abcam, Cambridge, UK) and goat anti-mouse immunoglobulin G (IgG) secondary antibody (Alexa Fluor 488; Thermo Fisher Scientific). Tetramethylrhodamine (TRITC-rhodamine; Thermo Fisher Scientific) and 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) were used to stain the cytoskeletons and nuclei, respectively. The stained coverslips were fixed on slides with sealant and kept in the dark. CLSM (Carl Zeiss AG, Baden Württemberg, Germany) was used to capture images of the cells under different light sources [23 (link)].