Data Availability StatementAll datasets generated for this research are contained in the content/supplementary materials. AIS. (DIV) with a complete of 0.8C1.6 g DNA pr. coverslip McMMAF (25 mm in size) using Lipofectamine2000 (Thermo Fischer Scientific) based on the manufacturers protocol. Neurons were left for expression for 48 h before either fixation or FRAP experiments were performed. Immunocytochemistry Neurons (10 DIV) were fixed in 4% paraformaldehyde in PBS for 20 min or, in the case of pan-Nav1 stainings, for 2 min in 2% paraformaldehyde in PBS followed by 10 min in 100% methanol at ?20C. Blocking of unspecific binding was performed for 30 min McMMAF with 0.2% fish skin gelatin (Sigma-Aldrich) in PBS for surface-staining or in 0.1% Triton X-100 (Sigma) in PBS (PBST) for total-staining for 30 min at room temperature (RT). Next, neurons were incubated with primary antibodies diluted in 0.2% fish skin gelatin in McMMAF PBS or PBST for 1 h at RT. Lastly the neurons were incubated with secondary antibodies diluted in 0.2% fish skin gelatin in PBS or PBST for 45 min at RT. Primary antibodies McMMAF used: mouse anti-CD4 (1:25C1:50 dilution; clone 18C46; Santa Cruz Biotechnology), mouse anti-CD4 (1:50 dilution, MT310, Santa Cruz Biotechnology), rabbit anti-MAP2 (1:100 dilution, H-300, Santa Cruz Biotechnology), mouse anti-pan-Nav1 (1:100 dilution, clone BPES1 N419/40, RRID:AB_2491098, Neuromab), mouse anti-ankG (1:5, clone N106/65, RRID: AB_10673449, Neuromab), mouse anti-GFP (1:5, clone 4C9, Developmental studies Hybridoma Bank). Primary antibodies were detected using AlexaFluor?-conjugated secondary antibodies (Thermo Fischer Scientific). Coverslips were mounted on microscope slides using ProLong Gold or Diamond Antifade Reagent (Thermo Fischer Scientific). Confocal Microscopy Laser-scanning confocal microscopy was performed on upright LSM710 or LSM780 microscopes from Zeiss equipped with argon and helium-neon lasers and a 63x, 1.4 numerical aperture, oil-immersion objective. Imaging settings included a pinhole of 1 1 airy unit and a pixel format of 1024 1024. Line averaging was used to reduce noise. Images in.lsm file format were processed using Zeiss ZEN Black and Blue 2011 software and exported in.tiff format for figures. Image Analysis Images in.lsm file format were analyzed using Fiji (Fiji_Is_Just_ImageJ). The AIS was identified using either pan-Nav or AnkG as marker. Three segmented lines were manually placed in each image; a 90 m segmented line starting from the soma was drawn along the axon, a 20 m segmented line was drawn in a dendrite projecting from the soma and a 10 m segmented line in a region with no cells was included for background subtraction. Fluorescence intensity profiles were extracted from each line and the mean background value subtracted. Mean AIS intensity (the proximal 0C30 m of the axonal line), mean distal axon intensity (60C90 m of axonal line) and mean dendrite intensity (0C20 m of dendrite line) were calculated followed by calculation of AIS/Dendrite and AIS/Distal axon ratios as previously described (Rasmussen et al., 2007). For quantifications of the AIS start and length of ankG immunolabeling, axonal profiles were smoothed using a 1.45 m sliding mean and the mean fluorescence intensity in the axonal region distal to the AIS (50C60 m of axonal line) was subtracted from each value along the axonal profile. AIS start and McMMAF end positions were identified as the points where fluorescence intensities increased above and dropped below 33% of the max axonal fluorescence intensity, respectively, in line with previous reports (Grubb and Burrone, 2010). AIS length was calculated as the difference between AIS start and AIS end. AIS density of ankG and pan-Nav1 immunolabeling was calculated.