4.8. Immunofluorescence Microscopy

hKC, JEB-KC and JEB∆6∆7 cells (1.5 × 10⁵) were seeded per well into 2-well chamber slides (Nalge Nunc International, Lab-Tek^® II Chamber Slide™ system, #154461, Rochester, NY, USA) and cultivated for 48 h in serum-free medium (CELLnTEC, #CnT-PR). At confluence, cells were fixed with ice-cold methanol for 10 min, washed with PBS and blocked with 5% BSA (Sigma, #A3294) in PBS for 1 h. The primary antibody against type XVII collagen (abcam, #ab184996) was incubated overnight at 4 °C. After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, #A11008, Waltham, MA, USA) and DAPI (Sigma Aldrich, #D9542) for 1.5 h at room temperature. Imaging was performed on a Zeiss LSM710 confocal microscope, and images were analyzed using ImageJ software.