Rabbit anti foxp2

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)].

Ten 30 micron sections were generated for each surgical group and were slide mounted and incubated in blocking solution as previously described (33 (link)). Sections were incubated overnight with rabbit anti-FOXP2 (1:200, abcam), mouse anti-NeuN (1:200, Millipore), and then incubated for 60 min with anti-rabbit and anti-mouse secondary antibody conjugated with a fluorophore (1:1000, FlourAlexa) and DAPI nuclear stain solution (1:1000, Invitrogen) as previously described in O’Keefe et al, 2013 (34 ). Images were obtained with an inverted light Zeiss-Axiovert fluorescence microscope.

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).