Alexa fluor 543 conjugated goat anti rabbit igg

For immunofluorescence, H3255 cell lines were grown on glass coverslips to 70% confluence. Cell cultures were washed with PBS and fixed with 4% (w/v) formaldehyde in PBS for 30 min at 37°C. After washing with PBS, cells were permeabilized with 0.1% v/v Triton X-100 in PBS for 10 min, rinsed three times in PBS and treated with 5% w/v bovine serum albumin for 1 h. Cells were then incubated overnight at 4°C with either rabbit anti-EGFR pAb (sc-03; Santa Cruz Biotechnology; 1:2,000), mouse anti-CRT mAb (BD Transduction Laboratories^™; 1:1,000) or mouse anti-B1-Integrin mAb (MAB2253; Millipore; 1:500) primary antibodies and for 1 h at room temperature with either Alexa Fluor 543-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse IgG (ThermoFisher Scientific; 1:1,000) secondary antibodies. All antibodies were diluted in PBS containing 5% w/v bovine serum albumin. Nuclei were stained using ToPro3 (Invitrogen). Confocal cellular images were captured with an inverted Zeiss LSM 510 Pascal laser confocal microscope (Carl Zeiss, Jenna, Germany), using a 63/1.4 Plan-Apochromat objective. Quantification of colocalization was performed using Image J Colormap Software.

For immunofluorescence, HCC827-GR cell lines were grown on glass coverslips to 80% confluence. Cell cultures were washed with PBS and fixed with 4% (w/v) formaldehyde in PBS for 30 min at 37°C. After washing with PBS, cells were permeabilized with 0.1% v/v Triton X-100 in PBS for 10 min, rinsed three times in PBS and treated with 5% w/v bovine serum albumin for 1 h. Cells were then incubated overnight at 4°C with mouse anti-EGFR pAb (sc-03; Santa Cruz Biotechnology; 1:800) and rabbit anti-MET mAb (Cell Signaling; 1:1,000) primary antibodies and for 1 h at room temperature with either Alexa Fluor 543-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse IgG (ThermoFisher Scientific; 1:1,000) secondary antibodies. All antibodies were diluted in PBS containing 5% w/v bovine serum albumin. Nuclei were stained using ToPro3 (Invitrogen). Confocal cellular images were captured with an inverted Zeiss LSM 510 Pascal laser confocal microscope (Carl Zeiss, Jenna, Germany), using a 63/1.4 Plan-Apochromat objective.