Goat anti vat1

Axonal staining and measurement were performed as previously described [20 (link), 23 (link)]. Briefly, monoclonal antibodies against phosphorylated neurofilament heavy protein (SMI31, 1:1000, Covance, Battle Creek, MI, USA) or class III beta-tubulin (TUJ1, 1:1000, Biolegend, San Diego, CA, USA) were used. For postnatal DRG neurons, the lengths of the 15 longest axons in each chamber were measured using a microscopic computer imaging device (MCID) system. The axonal length was recorded for 3 days from DIV3 to DIV5. For adult DRG neurons, one time point axonal length was measured on DIV3 according to a published protocol [28 (link)]. Immunostaining for DRG tissues was performed according to a published protocol [24 (link)]. Briefly, L3–L6 DRGs were isolated, fixed in 4% paraformaldehyde, and embedded in paraffin. The slices were cut in 6 μm thickness. The following primary antibodies were used: rabbit anti-FOXP2 (1:50, Abcam) and goat anti-VAT1 (1:50, Santa Cruz). Cellular nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1:10000, Thermo Fisher Scientific). A polyclonal antibody against protein gene product 9.5 (PGP 9.5, 1:1000; Millipore) was used to detect intraepidermal nerve fibers in plantar skin. The nerve fiber densities were calculated according to a published protocol [29 (link)].

Total proteins from cultured DRG neurons were isolated on DIV6 according to published protocols [25 (link), 27 (link)]. Total protein from DRG, sciatic nerve and foot pad tissues of db/db and db/m mice were extracted using the same methods. In vitro samples from 4 individual microfluidic chambers were pooled for one Western blot. The protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Western blot was performed according to previously described methods [25 (link), 27 (link)]. Briefly, equal amounts of proteins were loaded. Primary antibodies were rabbit anti-ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10, 1:1000, Abcam, Cambridge, UK), rabbit anti-DCX (doublecortin, 1:500, Abcam), rabbit anti-c-MET (tyrosine-protein kinase met, 1:500, Abcam), rabbit anti-FOXP2 (1:1000, Abcam), goat anti-NOTCH1 (1:1000, Santa Cruz), rabbit anti-ROCK1 (Rho associated coiled-coil containing protein kinase 1, 1:500, Abcam), rabbit anti-SYNJ1 (Synaptojanin 1, 1:1000, Sigma-Aldrich), rabbit anti-VAMP2 (vesicle-associated membrane protein 2, 1:1000, Cell Signaling Technology, Danvers, MA, USA), goat anti-VAT1 (1:1000, Santa Cruz), and mouse anti-β-actin (1:10000; Abcam). The optical density of protein bands was measured and calculated by means of Fluorchem E instrument (ProteinSimple, San Jose, CA, USA).