Fig. 2: Genetic code expansion and click labeling of NFL in live primary mouse cortical neurons (MCNs).
From: Minimal genetically encoded tags for fluorescent protein labeling in living neurons
a, c MCNs expressing NFLK363TAG-FLAG, NFM and NES PylRS/tRNACUAPyl in the presence of TCO*A-Lys. a Three days after transfection, neurons were labeled with SiR-tz via click chemistry, then fixed and stained with anti-FLAG antibody, followed by AF488-conjugated secondary antibody. After fixation and immunocytochemistry labeling, neurons were imaged on a confocal scanning microscope. b Colocalization analysis of SiR-tz and FLAG intensity in the region within the dashed box from panel a, with Pearson’s correlation coefficient (PCC) shown above the scatterplot. Fluorescence intensities in the graph are shown as absolute gray values of 16-bit depth images. c Three days after transfection, neurons were labeled with either BODIPY-tz, TAMRA-tz, or SiR-tz. Excess dye was washed for 3 h, and live neurons were imaged on a confocal scanning microscope. In TAMRA-tz panel, background staining of lysosomes is highlighted in both NFLK363TAG-expressing neuron (arrows) and in non-transfected neurons (arrowheads). Z-stack images are shown as maximum intensity projections. Scale bars: 20 µm (a, c). Data were collected from three independent experiments. Source data are provided as a Source Data file.
