Donkey anti rabbit 488

A549 cells (1,000 cells/cm²) were seeded overnight on coverslips. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature, followed by permeabilization using 0.1% Triton in PBS for 5 min and blocking of unspecific binding with 5% donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) in PBS. These steps were done at room temperature and washing with PBS was done between any steps. Primary antibodies were rabbit anti-early endosomal antigen (EEA)1 (1:300; cat. no. 610456; BD Bioscences, San Jose, CA, USA) and mouse anti-KRAS (1:100; cat. no. ab172949; Abcam, Cambridge, MA, USA) and were applied overnight at 4˚C. After additional washing steps, cells were incubated with secondary donkey anti-rabbit-488 (1:200; cat. no. 711-545-152l; Jackson ImmunoResearch Laboratories, Inc.) and anti-mouse-Cy3 (1:200; cat. no. 715-165-150; Jackson ImmunoResearch Laboratories, Inc.) antibodies in PBS with 5% donkey serum for 30 min at room temperature, followed by washing and addition of Hoechst 33342 Solution (1 µg/ml; Thermo Fisher Scientific, Inc.) for 3 min, followed by washing, H₂O for 3 min, 100% ethanol for 3 min and air-drying prior to embedding (all steps at room temperature). Images were captured with a Zeiss LSM 780 NLO confocal microscope and quantification was performed using ImageJ, version 1.51 (National Institute of Health, Bethesda, MD, USA).