Sp5c spectral confocal laser scanning microscope

For immunofluorescence (IF) staining slides were prepared as described above for IHC and stained with anti-IgG antibody (1:50, Sigma F8264) as described [23 (link)]. Images were captured on a Leica SP5C Spectral Confocal Laser Scanning Microscope.

The IgG internalization assay was performed to detect internalized DSG3 as previously described [27 (link),29 (link)]. Briefly, HaCaT cells were incubated with a monoclonal antibody (AK23) against the extracellular domain of DSG3 [41 (link)], in media containing 1.5 mM calcium for 30 minutes on ice. Cells were then washed and incubated with PV IgG (400 μg/ml) or normal human IgG (400 μg/ml) at 37° C for one hour to induce DSG3 internalization. Subsequently, cells were treated with acid wash solution (100 mM glycine, 20 mM magnesium acetate, 50 mM potassium chloride, pH 2.2) to remove surface-bound DSG3 antibody before fixation. For knockdown studies, cells were transfected with siRNA prior to calcium switch. Images were acquired by Leica SP5C Spectral confocal laser scanning microscope under the same color intensity threshold and analyzed using ImageJ. Quantification was done by counting green fluorescent puncta in randomly sampled microscopic fields with Analyze Particle, which was then normalized by the number of cells so that the net result reflects the average number of puncta (internalized DSG3) within one cell.

To examine the cellular uptake of NPs, 1 × 10³ cells/well were seeded in glass-bottom microwell dishes (MatTek Corporation) for 12 h and then incubated with PLGA-PEG or PLGA-PEG-TFA NPs at 37 °C for 24 h. The cells were then washed twice with PBS and fixed with 4% paraformaldehyde for 10 min. Cell membranes were then stained with Did (1:400) and the nuclei with DAPI (Invitrogen by Thermo Fisher Scientific). The cells and NPs were imaged using a Leica SP5C Spectral Confocal Laser Scanning Microscope. The NPs loaded FITC dye was used in this experiment instead of NIR dye.