GxTX-594 colocalizes with Kv2-GFP. (A) Fluorescence from CHO cells transfected with Kv2.1-GFP (top) or Kv2.2-GFP (bottom) and labeled with GxTX-594. Optical sections were near the glass-adhered cell surface. Cells were incubated with 100 nM GxTX-594 for 5 min and rinsed before imaging. Fluorescence shown corresponds to emission of GFP (left), GxTX-594 (middle), or as an overlay of GFP and GxTX-594 (right). Scale bars, 20 μm. (B) Pearson correlation coefficients for GxTX-594 colocalization with Kv2.1-GFP or Kv2.2-GFP. (C) Ratio of fluorescence intensity of the same cells in B when excited at 594 nm versus 488 nm from cells expressing Kv2.1-GFP or Kv2.2-GFP. In B and C, pixel intensities were background subtracted before analyses by subtracting the average fluorescence of a background ROI that did not contain cells from the ROI containing the cell. Circles represent measurements from individual cells, and each transfection group contains data from two separate applications of GxTX-594. Bars represent the mean. No significant difference was detected between Kv2.1-GFP or Kv2.2-GFP by Mann–Whitney U test (P = 0.74).