Cellular colocalization of TM601 and clathrin light chain in chlorpromazine treated U373 glioma cells. Immunocytochemistry of U373 glioma cells followed by confocal microscopy showed cellular localization of TM601 (red) and clathrin (green) in relation to the nucleus (blue) in TM601 treated control (A) and in TM601 and 10 μg/ml chlorpromazine treated cells (B, C). The chlorpromazine treatment resulted in accumulation of TM601 in the perinuclear region (B, C). The schematic illustration of chlorpromazine action on TM601 uptake is shown (E). The cellular colocalization of TM601 (red) and clathrin light chain (green) was detected by immunofluorescence using the Colocalization Tool (D and F) as described in the Methods. An overlay mask (white pixels) placed on each image visualizes the colocalization of pixels above the background threshold (D and F). It shows that more of white pixels were observed in TM601 and 10 μg/ml chlorpromazine treated (F) than in TM601 (D) only treated glioma cells. This is due to a higher level of TM601 in the presence of chlorpromazine resulting in higher red immunofluorescence which merged with green immunofluorescence derived from clathrin. The following three fluorescent color channels were used: red (TM601), green (clathrin light chain), and yellow (merge of red and green fluorescence) to determine the extent of colocalization of TM601 and clathrin light chain by confocal microscopy. The merged red and green fluorescence in glioma cells (C) was compared to the controls of TM601 (red insert in C) alone or clathrin light chain (green insert in C) alone. Data are representative of total n = 3 experiments. The Colocalization Tool in D and F was used in one out of 3 experiments evaluating total of 15-26 cells in 3 fields. Scale bars = 10 μm, magnification 3780 ×.