Prolong gold antifade dapi

The HaCaT cells were cultured on glass coverslips in 24-well plates (2 × 10⁴/well) for 24 h. Then, co-culture with C. acnes and the treatment with the leaf extract or castalagin were conducted as previously described, for 48 h. At the end of treatment, the cells were fixed using PBS 1×/PFA 4% solution for 15 min, and then, they were washed with PBS 1X. The blocking–permeabilizing solution was added (BSA 5%, Triton-X 0.3%, in PBS 1X) for 1 h, and then, immunostaining with mouse anti-human CK10 (3C2F5, NBP2-61736) (1 μg/mL) (Novus biologicals, Milan, Italy) was performed as previously described [20 (link)]. After overnight incubation at 4 °C, the AlexaFluor^® 488-conjugated anti-mouse antibody #150113 (Abcam, Cambridge, CB2 0AX, UK) was added for 1 h, and then, the cells were washed three times and mounted on glass slides with ProLong Gold Antifade DAPI (Cell Signaling, MA, USA). The fluorescent images were acquired through the use of confocal microscopy (LSM 900, Zeiss, Oberkochen, Germany).

HaCaT cells were cultured on glass coverslips in 24-well plates (3 × 10⁴/well) for 9 days. Then, cells were treated for 24 h with IL-4 (100 ng/mL) and HVE (5 and 50 μg/mL) or HT (1 and 5 μM). Cells were fixed via incubation with formaldehyde (4%) for 15 min, and then they were washed 3 times with PBS 1X. The blocking-permeabilizing solution was added (BSA 5%, Triton-X 0.3%, in PBS 1X) for 1 h, and then immunostaining with anti-K10 (3C2F5) (1 μg/mL) and anti-IVN (LHK1) (2 μg/mL) mouse antibodies (Novus biologicals, Milan, Italy) was performed at 4 °C overnight. The day after, the AlexaFluor^® 488-conjugated anti-mouse antibody (Cell Signaling, MA, USA) was added for 1 h, and then cells were washed 3 times and mounted on glass slides with ProLong Gold Antifade DAPI (Cell Signaling, MA, USA). The fluorescent images were acquired using confocal microscopy (LSM 900, Zeiss, Oberkochen, Germany).