Immunocytochemistry was conducted to assess the expression and co-localization of EGFR and CD44 in MDA-MB-231 cells. Cells in Falcon® chambered cell culture slides (BD Biosciences) were fixed with 4% paraformaldehyde, washed with PBS, and incubated with 0.2% Triton X-100 for 13 min. Primary antibodies were applied to the cells in antibody-diluent (Dako, Denmark) were incubated overnight at 4°C. For secondary antibody reactions, Alexa Fluor®-488 or -594 conjugated secondary antibodies (Thermo Fisher Scientific) were used for staining and then mounted with ProLong® Gold Antifade Reagent with DAPI (Thermo Fisher Scientific). Imaging of the cells was performed using a Carl Zeiss confocal microscope (Weimar, Germany), and the intensity of the EGFR and CD44 signal was analyzed using the intensity profile tool.
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