P65 cst 8242s

The plates were washed in PBS, fixed for 10 min with 4% paraformaldehyde (pH 7.4), and permeabilized for 20 min with 0.5% Triton X-100 at room temperature. After washing with PBS, 5% FBS was added to the center of the dish and allowed to stand at 20–37 °C temperature for 30 min. The blocking solution was then removed, a sufficient dilution of primary antibody p65 (cst-8242s, 1:200; Cell Signaling Technology, Danvers, MA, USA) was added, and the cells were incubated at 4 °C overnight. A fluorescence-conjugated secondary antibody was added. After washing with PBST, the excess liquid was absorbed by absorbent paper with diluted fluorescent secondary antibody, incubated at 20–37 °C for 1 h, and soaked in PBS for 3 min each time. These steps were performed in the dark as much as possible. Finally, nuclei were counterstained with an anti-fluorescence quencher containing DAPI, and images were taken via a fluorescence microscope.

Immunofluorescence staining was conducted according to standard procedures. Paraffin sections of tissue or cell climbing sheets were incubated with specific primary antibodies (GCA, PA5-77127, Invitrogen,1:200; F4/80, ab6640, abcam,1:200; PHB2, sc-133094, Santa Cruz,1:200; P65, CST8242S, Cell Signaling Technology,1:400, Ly6g/6c,Biolegend,108403,1:200;Perilipin-1,Cell Signaling Technology,9349 S,1:200) at 4 °C overnight. After washing with PBS for 3 times, the sections or sheets were incubated with corresponding fluorescent secondary antibodies. The cell nuclei were labeled with DAPI. The cytoplasmic membrane was stained with Dil. The results were imaged by fluorescence microscope or confocal microscopy.