Imager z1

A wide-field fluorescence microscope (Imager Z1/Zeiss, www.zeiss.com) in combination with TissueFAXS software (TissueGnostic GmbH, www.tissuegnostic.com) was used to view overall cell performance after labeling of cells. Binding and uptake of markers as well as colocalization of proteins in single cells was investigated using a Zeiss Axiovert 200 confocal microscope equipped with a 63× oil immersion lens (Zeiss). Pictures were digitalized using Volocity software (Perkin Elmer, www.perkinelmer.com). Averages of 60–100 cells per experiment were evaluated. The Pearson correlation coefficient value (PCC), r, was used as a statistic measure to show colocalization of two proteins. The values for r were considered as very weak (0.0–0.19), weak (0.20–0.39), moderate (0.40–0.59), strong (0.60–0.79) and very strong (0.80–1.0) (Williams, 1996 (link); Dunn et al., 2011 (link)). Images were processed using Image J and Adobe Photoshop software.

Melanoma cells were plated into 24-well cultivation plates with glass coverslips and were allowed to attach overnight. Cells were exposed to 1,000 nM PLX4032 for up to 7 days. At specific time points, indicated in the results, cells were washed with 1X PBS and fixed with 4% paraformaldehyde solution in 1X PBS for 30 min at room temperature (RT). The slides were washed with 1X PBS and the cells were blocked and permeabilized in blocking buffer (1X PBS containing 5% goat serum and 0.5% saponine) for 1 h at RT. Cells were incubated for 1 h at RT with anti-CSPG4 antibody 9.2.27, diluted 1:1,000 in blocking buffer, washed three times with 1X PBS and incubated for 1 h at RT with goat anti-mouse secondary IgG antibodies Alexa Fluor 568^®, diluted 1:2,000 in blocking buffer. Finally, nuclei of the cells were counterstained with DAPI (1:10,000), slides were washed three times with 1X PBS and mounted on a glass slide using Fluoromount-G (Thermo Fisher Scientific, Inc.). A wide-field fluorescence microscope (Imager Z1/Zeiss) in combination with TissueFAXS software version 4.2.6245.1019 (TissueGnostic GmbH) was used to examine the slides.