Mix n stain cf640r

Rabbit anti-LAMTOR4 (cat. 13140) (1:200) was from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-α-tubulin (DM1A; cat. T6199) (1:1,000) and D-biotin (cat. 47868) were from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-TfR (cat. ab84036) (1:200) and rabbit anti-PMP70 (cat. ab3421) (1:500) were from Abcam (Cambridge, UK). Rabbit anti-Sec31A (cat. 17913-1-AP) (1:500) was from Proteintech (Rosemont, IL, USA). Mouse anti-calnexin (clone C8.B6; cat. MAB3126) (1:1,000) was from EMD Millipore (Burlington, MA, USA). Mouse anti-pan-neurofascin (external) (clone A12/18; cat. 75–172) (1:50) was from the University of California, Davis/NIH NeuroMab Facility (Davis, CA, USA). Mix-n-Stain-CF640R (cat. 92245) was from Biotium (Fremont, CA, USA) and was used to label the antibody against neurofascin. Mouse anti-LAMP1 (H4A3) (1:500) was from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA). Mouse anti-EEA1 (cat. 610457) (1:1,000) was from BD Biosciences (San Jose, CA, USA). All secondary antibodies (donkey anti-rabbit and anti-mouse conjugated with Alexa Fluor 488, 555, or 647) (1:1,000) were from Invitrogen (Carlsbad, CA, USA). NeutrAvidin Protein (cat. 31050), LysoTracker Blue DND-22 (cat. L7525), AF647-dextran (cat. D22914), MitoTracker Red CMXRos (cat. M7512), and DQ Red BSA (cat. D12051) were from Thermo Fisher Scientific (Waltham, MA, USA).

Labelling of the surface channel was performed before imaging. Neurons were rinsed with NIS, to remove the Neurobasal media. For experiments with CD4, cells were incubated for 10 min at 37 °C with a monoclonal antibody against CD4 (MABF573, Millipore, Billerica, MA), which we had previously directly conjugated with a CF640R fluorophore (antibody labelling kit Mix-n-stain CF640R, Biotium, Hayward, CA), diluted 1:1000 in NIS. For experiments using the Nav1.6 and Kv1.4 constructs containing the extracellular BAD, cells were incubated for 10 min with streptavidin-conjugated CF640R (Biotium, Hayward, CA) diluted 1:1000 in NIS. Both streptavidin-CF64R and Anti-CD4 labelling was done at 37 °C in the presence of bovine serum albumin (cat. A0281, Sigma, St Louis, MO). Excess label was removed by rinsing with neuronal imaging saline.