Goat anti rabbit igg h l conjugated to alexa fluora 488 secondary antibodies

For in vitro immunofluorescence assay, HaCaT cells (1 × 10⁵ cells/ml) were grown to 50%–70% confluency in 8‐well chambers (ibidi,). Media was replaced with media plus 4‐PP at 12.5, 25 and 50 μM for l h. After UVB irradiation (0.03 J/cm²), cells were incubated for 15 min.
For in vivo immunofluorescence assay, mouse skins separated from back were embedded with OCT solution (Leica Biosystems Richmond Inc.,) in frozen section. Frozen section was cut into 6‐μm‐thick sections under a microscope (Cryostat CM1850, Leica Biosystems,) and then attached to microscope slides (Thermo Fisher Scientific,).
Cells and frozen tissues were fixed with 4% formaldehyde and permeabilized with ice‐cold 100% methanol. After blocking, cells were incubated with specific antibodies at 4°C overnight. Goat anti‐rabbit IgG H&L conjugated to Alexa Fluora® 488 secondary antibodies (Abcam) were incubated with the cells for 1–2 h. Nuclei were counterstained with DAPI antibody (VECTASHIELD®: Vector Laboratories, Burlingame, CA). The location of c‐Jun was determined using a confocal laser scanning microscope (Carl Zeiss Co Ltd.,).

RAW 264.7 cells were seeded (5 × 10⁴ cells/ml) in an 8-well chamber (ibidi, Germany) and allowed to reach 50-70% confluency. The media were then substituted with media containing varying amounts of SGE (25, 50, and 100 μg/ml). After incubating for 1 h, LPS (1 μg/ml) was added to the media, and the cells were incubated for 5 min. After incubation, the cells were fixed with 4% formaldehyde and permeabilized with ice-cold 100% MeOH. After blocking, cells were incubated with anti-p65 antibody (VECTASHIELD: Vector Laboratories, USA) overnight at 4°C. Goat anti-rabbit IgG H&L conjugated to Alexa Fluora 488 secondary antibodies (Abcam) were incubated with the cells for 1-2 h. The nuclei were counterstained with DAPI. The location of NF-κB p65 was determined using a confocal laser scanning microscope (Carl Zeiss Co. Ltd., Germany).