On DIV 14, neurons were fixed with room temperature 2% PFA in 0.1 M PBS and washed two times with 1× PBS, and coverslips were mounted on microscope slides (Fisher Scientific, catalog no. 12-550-15) using Vectashield mounting media (Vector Labs, catalog no. H1000). A blinded experimenter performed all imaging. Images were captured on a Nikon (Tokyo, Japan) Eclipse Ni upright microscope, using a Nikon Intensilight and Photometrics Coolsnap HQ2 camera to image Lifeact-GFP. Images were captured with Nikon Elements 4.20.02 image capture software using 60× oil immersion objective (NA, 1.40; Nikon Plan Apo). Z series images were acquired at 0.15-μm increments through the entire visible dendrite. Dendrites were selected for imaging by using the following criteria: (i) minimum of 25 μm from the soma, (ii) no overlap with other branches, and (iii) must be a secondary dendritic branch. Before analysis, captured images were deconvolved using Huygens Deconvolution System (16.05, Scientific Volume Imaging, The Netherlands) with the following settings: CMLE; maximum iterations, 50; signal-to-noise ratio, 40; and quality, 0.01. Deconvolved images were saved in .tif format.

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