Goat anti rabbit igg fitc

To analyze the endogenous Zscan4 expression profile (percentage of Zscan4⁺ cells and fluorescence intensity), ESCs were collected and washed with cold PBS, then fixed in cold 4% PFA, permeabilized in 0.1% Triton X-100 in blocking solution (4% BSA in PBS) for 30 min, washed three times, and left in the blocking solution for 1 h. ESCs were incubated 1 h at room temperature with the primary antibody against Zscan4 (1:1000, Millipore), washed three times, and incubated for 30 min with secondary antibodies, FITC goat anti-rabbit IgG (1:1000, Beyotime). Samples were washed three times with PBS and the FACS analysis was performed using a FACSAria Flow Cytometer (BD Biosciences).

Raw 264.7 macrophages (5 × 10⁴/well) were seeded in a 3.5 cm glass bottom plate and treated with LPS + FLs for 12 h. Subsequently, cells were fixed with paraformaldehyde solution (4%) at atmospheric temperature for 30 min and then blocked with immunostaining blocking buffer (Beyotime, Shanghai, China) for 45 min. Cells were incubated with the monoclonal antibody p-P65 (1:100, Cell Signaling Technology, Boston, MA, USA) overnight at 4 °C. FITC-goat anti-rabbit IgG (1:1000, Beyotime, Shanghai, China) stained cells for 1 h, avoiding light. 4′,6-Diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) was added to each plate and incubated for 5 min. Finally, cells were observed under a confocal laser scanning microscope (CLSM, LSM880 Airyscan, Zeiss, Jena, Germany) with a 10× objective lens with NA 0.45 and a 63× oil objective lens with NA 1.4.