Immunofluorescence Staining Protocol

Immunofluorescence staining was performed as described previously [56 (link)] with some modifications. Cells were fixed with 1% paraformaldehyde (PFA) (Sigma-Aldrich) solution at room temperature for 15 min and permeabilized with 0.1% Triton X-100 (Merck, Darmstadt, Germany) for 10 min. After several washes with 1 × PBS, the fixed cells were blocked using 3% bovine serum albumin (BSA; Bovogen Biologicals, VIC, Australia) and 5% FBS, and subsequently incubated with indicated monoclonal antibodies (1:100) at 4 °C overnight. Cells were washed thrice with PBS and incubated with the cyanine 3 (Cy3)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. Samples were counterstained with 100 μL DAPI. Finally, cells were mounted and observed using a fluorescent or FV10i confocal microscope (Olympus, Center Valley, PA, USA).