Immunofluorescence analysis of desmosome proteins

HaCaT cell line was seeded on glass coverslips with cell culture chambers (Nun Lab-Tek II Chamber Slide System, Thermo Fisher Scientific, Waltham, MA, USA) and cultured for at least 2 days in DMEM GlutaMAX medium containing 1 mM CaCl₂ and 10% FCS and grown to confluence. Cells were treated with either IgG from PV patients’ or HD serum for 20 h in DMEM GlutaMAX medium containing 1 mM CaCl₂ without FCS. After removing the medium and washing with PBS 1× complemented with CaCl₂ and MgCl₂ (Eurobio Scientific, Les Ulis, France), the cells were then fixed with ethanol 100% for 10 min at RT. The fixed cells were rinsed and permeabilized with Triton X-100 at 0.3% for 10 min (Sigma). After washing, cells were blocked for 30 min with 1% rat serum in PBS 1× at RT. Fluorescent-labeled antibodies staining DSG3 and flotillin-2, a protein associated with lipid microdomains that interact with desmosome proteins [28 (link)], were incubated for 1.5 h in the dark at RT in PBS 1× containing 1% BSA. Finally, coverslips were mounted with ProLong^™ Diamond Antifade Mountant containing DAPI (Thermo Fisher Scientific, Waltham, MA, USA). All samples were analyzed with a Leica SP8-UV confocal microscope.