Immunostaining of Keratin 10 in C. acnes-treated HaCaT Cells

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).