Rabbit anti phospho p65 antibody

For immunofluorescence assay, J774A.1 cells (3 × 10⁵/well) were seeded on coverslips in 12 well plate and treated with Cs or CsB (150–50 µg/mL) and LPS (1 µg/mL), as described above, for 20 min in order to detect NF-kB nuclear translocation.
Cells were then fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.1% saponin in PBS for 15 min. After blocking with BSA and PBS for 1 h, macrophages were incubated with Rabbit anti-phospho p65 antibody (Santa Cruz Biotechnologies, Dallas, TX, USA) for 1 h at room temperature. The slides were then washed with PBS for three times and fluorescein-conjugated secondary antibody (FITC) was added for 1 h, DAPI was used for counterstaining of nuclei. Coverslips were finally mounted in mounting medium and fluorescent images were taken under the Laser Confocal Microscope (Leica TCS SP5, Wetzlar, Germany) [40 (link)].

For immunofluorescence assay, J774A.1 cells (3 × 10⁵/well) were seeded on coverslips in a 12 well plate and treated with LFE (25 μg mL⁻¹) for 1 h and then simultaneously to LPS (1 μg mL⁻¹) for 20 min in order to detect Nuclear Factor-kB (NF-kB) nuclear translocation and for 1 h in order to detect Nuclear Factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation. Cells were then fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min and permeabilized with 0.1% saponin in PBS for 15 min. After blocking with bovine serum albumin (BSA) and PBS for 1 h, macrophages were incubated with Rabbit anti-phospho p65 antibody (Santa Cruz Biotechnologies, Dallas, TX, USA) or anti-Nrf2 antibody (Santa Cruz Biotechnologies) for 1 h at room temperature. The slides were then washed with PBS for three times and fluorescein-conjugated secondary antibody (FITC) was added for 1 h, 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) was used for counterstaining of nuclei. Coverslips were finally mounted in mounting medium and fluorescent images were taken under the Laser Confocal Microscope (Leica TCS SP5, Leica, Wetzalar, Germany) [20 (link)].