Time-resolved colocalization studies of S100A4 and MYH9 in activated NK cells. Time-resolved colocalization studies of S100A4 and MYH9 in activated NK cells. NK cells were incubated and activated with K562 target cells (‘T’ for target cell and ”NK” for NK cell) for 5, 10, 15, 20, and 30 min. First two rows show proof of concept colocalization events between Perforin (red) and CD107a (green), and between Perforin (red) and MYH9 (green). Time-resolved activation of NK cells and stain for S100A4 (red) and MYH9 (green) and DNA (DAPI, blue) with appropriate antibodies revealed remarkable time-dependent formation of colocalization events at the NKIS. Anti S100A4 was stained with goat anti-mouse IgG (Alexa 594) and anti-MYH9 (Myosin IIA) with goat anti-rabbit IgG (Alexa 488) supplemented with DAPI (1:1000). Imaging was performed on an inverted microscope (Axiovert 100TV; Carl Zeiss, Jena, Germany) microscope using standard epifluorescence illumination (light source HXP120, Zeiss) and 63×/NA1.4 or 100×/NA1.4 plan-apochromatic objectives. Imaging was performed at room temperature with immersion oil. Images were acquired with a back-illuminated, cooled charge-coupled-device camera (CoolSNAP HQ2, Photometrics, Tucson, AZ, USA) driven by Metamorph software (Version 7.5.3.0; Molecular Devices Corp., Downingtown, PA, USA). Five independent experiments were performed and 20–30 images were acquired per coverslip in each experiment. Image analysis was performed with ImageJ (version 1.44p). Maxi Scheiter et al. Mol Cell Proteomics 2013;12:1099-1114 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.