Fig. 3.
2C-Fab-GFP specificity. (A) HEK293 cells were divided into two groups and were separately transfected with plasmid containing mCherry-tagged 5-HT2C (5-HT2C/mCherry, red) or cyan fluorescent protein (CFP)–tagged 5-HT2A (5-HT2A/CFP, blue). Following transfection, the cells were mixed and plated on a single coverslip and stained live with 2C-Fab-GFP (green) for 30 minutes at room temperature. White scale bar = 10 µm. The merged image (right panel) shows 2C-Fab-GFP plasma membrane labeling of cells expressing 5-HT2C/mCherry (shown as yellow) and no labeling of cells expressing the most closely related protein in the genome, the 5-HT2A receptor (no overlap of green and blue). (B) Native 5-HT2C receptors endogenously expressed in choroid plexus epithelial cells labeled with 2C-Fab-GFP (green) are shown in the left panel. The same field of cells costained with an antibody to transthyretin (red) is shown in the middle panel. The merged image is shown in the right panel. White scale bar = 50 µm. (C) Optical sections (0.5 µm thick) from a Z-stack (6.0 µm) showing the apical surface, lateral plasma membrane, and basolateral regions of cells costained with 2C-Fab-GFP and transthyretin. The relative position within the Z-stack is noted in the lower-left corner of each image. (D) Choroid plexus epithelial cells labeled with the 2C-Fab-GFP probe in the presence of 1 μM 5-HT. The left panel shows an entire field of cells (white scale bar = 50 µm). The middle panel shows a single cell at higher magnification (white scale bar = 50 µm). The right panel shows the differential interference contrast (DIC) overlay of the cell in the middle panel. The white arrows point to the lateral plasma membrane where there is little fluorescence, in contrast to cells cultured in the absence of 5-HT (Fig. 3C, middle panel).