Images were recorded using an inverted wide-field microscope (Olympus IX-71) equipped with a water-immersion objective lens [UPlanSApo, ×60 magnification, numerical aperture (NA) 1.2, Olympus]. Violet (emission maximum at 390 nm), cyan (emission maximum at 475 nm), yellow (emission maximum at 603 nm), and red (emission maximum at 634 nm) light-emitting diodes (LEDs) (Spectra X light engine, Lumencor) were used for excitation of DAPI, Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 647, respectively. The fluorescence light was separated from the LED light with a dichroic beam splitter (59004bs, Chroma Technologies) and additional excitation filters (BLP01-405R, Semrock, for DAPI; FF01-520/35, Semrock, for Alexa Fluor 488; BLP01-594R, Semrock, for Alexa Fluor 568; and BLP01-635R, Semrock, for Alexa Fluor 647) before being imaged on an electron-multiplying charge-coupled device (EMCCD) camera (DU-897-CS0-BV, Andor) with an effective pixel size of 160 nm; the temperature of the detector is −50°C.

Images were analyzed using the image line profile operation (with a line width of 5 pixels) Igor Pro (WaveMetrics). Units refer to CCD pixel readout under strictly constant settings and parameters for immunostaining and imaging protocols within a dataset. AUs refer solely to the fact that the CCD’s readout magnitude is arbitrary overall, but not within or between measurements. Specifically, it is not possible to compare fluorescence intensity units between different antibodies or detection protocols (STORM and widefield), but as the confidence intervals on repeated measurements with identical antibody batches and identical staining and imaging protocols show, there is good comparability within those measurements (fig. S3, B and C). Part of the imaging process is selection of suitable samples. We only used images from cells, where the entirety of the quantified axonal section is in the same focal plane. If the axon grows on top of other structures, the cell was rejected. Replication numbers for individual cells, cultures derived from individual embryos, and the number of litters were given in the figure legends where applicable.

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