Fig. 3. Pulse–chase click labeling of two NFL populations in live primary mouse cortical neurons (MCNs).
a A schematic representation of the experimental workflow. Eight days after plating, MCNs were transfected with NFLK363TAG-FLAG, NFM, and NES PylRS/tRNACUAPyl constructs. After 2 days of incubation with TCO*A-Lys, neurons were labeled with the first dye (BODIPY-tz), incubated with TCO*A-Lys for a further 2 days, and labeled with the second dye (SiR-tz). After the second labeling step, neurons were either fixed, stained with anti-FLAG antibody followed by AF555-conjugated secondary antibody, and imaged on a confocal scanning microscope (b), or live neurons were imaged on a confocal scanning microscope (c). Z-stack images are shown as maximum intensity projections. d MCNs expressing NFLK363TAG-FLAG, NFM, and NES PylRS/tRNACUAPyl, labeled with a modified dual-color labeling approach. MCNs were transfected and labeled with the first tetrazine dye in the same way as in a–c. After the first dye labeling, neurons were incubated with TCO*A-Lys for 3 h, labeled with the second dye, fixed, and immunostained. Single-plane images were acquired on a confocal scanning microscope. Arrows indicate chimeric neurofilaments composed of SiR-tz and BODIPY-tz labeled NFL segments. Scale bars: 20 µm (b–d), 10 µm (inset in d). Data were collected from three independent experiments. Scheme in panel a was partially created with BioRender.com.