The culture conditions and time frames for immunohistochemistry were identical to those used for flow cytometry except that the cells were grown on 0.7 cm2 eight-chamber slides (Becton Dickinson, Franklin Lakes, NJ) and were transfected with 30 µl transfection mix. Washes were performed by adding 500 µl PBS to each chamber, followed by 5 min at room temperature. The secondary antibody for microscopy was donkey anti-mouse conjugated with Alexa Fluor 568 (Molecular Probes, Eigene, OR). Image analysis of fluorescence microscopy photographs was performed using ImageJ [45 ] using the following procedure: Fluorescence intensity thresholds for the GFP signal were selected using wild-type images, to limit the regions of interest to cells that were actively producing the RHO-GFP fusion product. The selected areas for each GFP image were then applied to the AF568 antibody image from the equivalent view field. Background grayscale measurements were subtracted from the grayscale measurements of the GFP+ cells, before the red/green ratio was determined.
Confocal microscopy was performed using a Nikon Ti-E inverted microscope with a Nikon A1Si spectral detector. Cells analyzed with confocal microscopy were labeled with biotinylated antimouse CD44 (Biolegend, San Diego, CA) and streptavidin conjugated to Alexa Fluor 594.
Confocal microscopy was performed using a Nikon Ti-E inverted microscope with a Nikon A1Si spectral detector. Cells analyzed with confocal microscopy were labeled with biotinylated antimouse CD44 (Biolegend, San Diego, CA) and streptavidin conjugated to Alexa Fluor 594.