Ti lapp illumination system

Adult mouse DRG neurons transduced with AAV‐PHP.S (Chan et al, 2017 (link)) encoding HA‐Dendra2‐nucleolin (full length and ΔGAR), or N2a or HEK‐293 cells transfected with HA‐Dendra2‐nucleolin (full length and ΔGAR) expression plasmids were live imaged with the Nikon Ti‐LAPP illumination system equipped with a temperature‐controlled chamber and an Andor EMCCD camera. Cells were incubated with 10 µM Hoechst 33342 in medium for 10 min prior acquisition. Images were acquired in DAPI, GFP, TRITC, and DIC channels at ×60 magnification with Nikon CFI plan apochromat 60× oil lambda objective. Medium was removed, and the cells were immediately refocused and imaged or the t = 0 image, followed by careful addition of 1 ml ddH2O by pipetting and image acquisition using a same setting with 42 s interval for 5 min. Images were analyzed by NIS‐Elements software (Nikon).

Adult mouse DRG neurons transfected by Amaxa Nucleofector II with HA‐Dendra2‐GAR(WT) or with HA‐Dendra2‐GAR(N) expression plasmids were live imaged with the Nikon Ti‐LAPP illumination system equipped with a temperature‐controlled chamber, Digital Mirror Device (DMD), and an Andor EMCCD camera. Cells were grown 48 h in complete F12 medium prior acquisition. Images were acquired in GFP, TRITC, and DIC channels at ×60 magnification with Nikon CFI plan apochromat 60× oil lambda objective. The photoconversion was performed in the cell body area by DAPI channel (50% of maximum led intensity for 20 s) using DMD module. The images were collected using GFP and TRITC channels at 10‐s interval for 20 min. DIC channel was used prior to photoconversion and in last four time points. Images were analyzed by NIS‐Elements software (Nikon). A neurite segment located at a distance of 60–90 µm from the cell body was selected for the kinetic intensity analysis.