Immunocytochemistry stainings

For immunocytochemistry (ICC) stainings, ND7/23 cells were washed briefly with 0.01 M phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, pH 7.4). Tris-buffered saline (TBS; 20 mM Tris, 150 mM NaCl, pH 7.6) was used instead of PBS for immunostaining of neurofilament light chain (NfL) because of potential influence of PBS on phosphorylated neurofilaments. Afterwards, cells were fixed with 2% paraformaldehyde (PFA; Sigma Aldrich, cat. no. 158127) at RT. For labelling of tubulin β3, cells were fixed as described previously¹³.

After fixation, cells were briefly washed again, blocked and permeabilized. Following primary antibodies were used: anti-tRFP (turbo red fluorescent protein) antibody (Evrogene, cat. no. AB233), mouse anti-neurofilament 70 kDa antibody, clone DA2 (Merck Millipore, cat. no. MAB1615), AF647-conjugated anti-Tubulin β3 (TUBB3) antibody (BioLegend, cat. no. 801210), AF488-conjugated anti-TUBB3 antibody (BioLegend, cat. no. 801203), anti-TUBB3 antibody (BioLegend, cat. no. 801202). For actin labelling, cells were incubated with phalloidin AF647 (Thermo Fisher Scientific, cat. no. A22287).

The following secondary antibodies were used: goat-anti rabbit AF647 (Thermo Fisher Scientific, cat. no. A-21245), goat anti-rabbit AF647 Plus (Thermo Fisher Scientific, cat. no. A32733), goat anti-rabbit AF488 (Thermo Fisher Scientific, cat. no. A-11034), goat anti-mouse AF647 Plus (Thermo Fisher Scientific, cat. no. A32728), goat anti-mouse AF488 Plus (Thermo Fisher Scientific, cat. no. A32723), goat anti-mouse AF647 (Thermo Fisher Scientific, cat. no. A21236), goat anti-mouse AF555 (Thermo Fisher Scientific, cat. no. A21424), goat anti-mouse AF633 (Thermo Fisher Scientific, cat. no. A21052).

MCN were fixed on various days in vitro (DIV) with 4% EM grade PFA (Electron Microscopy Sciences, cat. no. 15710) diluted in PEM buffer (80 mM PIPES, 2 mM MgCl₂, 5 mM EGTA, pH 6.8) for 15 minutes at RT. After fixation, background fluorescence was quenched with sodium borohydride (Sigma Aldrich, cat. no. 71320), cells were washed 3 times (10 minutes each wash) with PBS, blocked and permeabilized. The following primary antibodies were used: mouse monoclonal anti-pan sodium channel antibody (panNav; Sigma Aldrich, cat. no. S8809), mouse monoclonal anti-ankyrin G antibody (Santa Cruz, cat. no. sc-12719) and mouse monoclonal anti-beta-spectrin II (βII spectrin) clone 42 (BD Biosciences, cat. no. 612 563). Goat anti-mouse secondary antibodies conjugated with AF647 Plus or AF647 were used.

Details on ICC staining steps and antibodies used in each figure are provided in Supplementary tables ^{S1, S2} and ^S3.

After labelling, ND7/23 cells and MCN were washed 3 times (5 minutes each wash) and imaged on the same day. Cells stained with anti-tRFP (NLS-mCherry transfected cells) were also imaged on the following day.