Dylight 649 tomato lectin

Tissue sections were blocked for 15 min in True Black Supressor (Biotium, Fremont, CA, USA) followed by 15 min in Carbo Free Blocking Solution (Vector Laboratories, Newark, CA, USA) with 0.3% Tx-100. Sections were then incubated overnight at 4 °C in Carbo Free w/0.3% Tx-100 and rabbit anti-PDGFR-β (1:50; Thermo Fisher, Waltham, MA USA #MA5-15143), Alexa 488-Milli-Mark^® FluoroPan Neuronal Marker (1:100; Millipore Sigma #MAB2300X), Cy3-GFAP (1:100; Millipore Sigma, St. Louis, MO, USA #C9205), and DyLight 649-tomato lectin (1:1000; Vector Laboratories, Newark, CA, USA #DL-1178-1). Sections were then incubated in Carbo Free with 0.3% Tx-100 and Alexa 405 Plus goat anti-rabbit (1:200; Thermo Fisher # A48254) for 1 h at room temperature and coverslipped in ProLong Diamond (Thermo Fisher # P36970). When labeling Aquaporin-4 instead of GFAP, Alexa 488-Aquaporin-4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA # sc-32739 AF488) and the Milli-Mark Pan Neuronal Marker antibody was labeled with CF555 Mix-n-stain labeling kit (Biotium) according to the manufacturer’s instructions. A list of labeling reagents used in this study can be found in Table 1.

Human insulin (Humulin R) was purchased from Eli Lily. FITC-insulin was purchased from Sigma-Aldrich. Vectastain Universal Elite ABC kit, Carbo-Free blocking solution, and DyLight 649-tomato lectin were purchased from Vector Laboratories. Bovine serum albumin (BSA) was purchased from Rockland Immunochemicals. Phospho-insulin receptor beta (Y1185) antibody was purchased from Bioss (#bs-5453R). Rabbit anti-insulin antibody and horseradish peroxidase conjugated anti-rabbit IgG antibody was purchased from Cell Signaling Technology. Neuro-Chrom pan-neuronal antibody was purchased from Millipore (#ABN2300). Phosphatase inhibitor cocktail was purchased from Research Products International. Complete mini protease inhibitor cocktail was purchased from Roche. SuperBlock blocking buffer, 4,6-diamidino-2-phenylindole (DAPI), IP lysis buffer, ProLong Diamond, and Alexa Fluor 568 goat anti-mouse antibody were purchased from Thermo Fisher. Any KD TGX gels, XT sample buffer, reducing agent, and Clarity Western ECL reagent were purchased from Bio-Rad. All other reagents were purchased from Sigma-Aldrich unless noted.

Brain sections fixed in PFA were incubated for 30 min in Carbo-Free blocking solution followed by 30 min in DyLight 649-Tomato lectin (1:1000, Vector Labs), washed and then coverslipped in ProLong Diamond for confocal microscopy. Trigeminal nerves fixed in PFA were blocked in PBS with 0.3% Tx-100 + 5% BSA and incubated in Neuro-Chrom primary antibody (1:1000) overnight at 4 °C. Sections were then washed and incubated in Alexa Fluor 568 goat anti-mouse secondary antibody (1:500) for 1 h at room temperature. Sections were washed and nuclei were stained with DAPI. Sections were then coverslipped in ProLong Diamond for confocal microscopy. Brain sections fixed in HistoChoice were blocked in PBS with 0.05% Tween-20 + 1% BSA for 1 h and then incubated in rabbit anti-insulin antibody (1:50) overnight at 4 °C. Standard ABC methods following the manufacturer’s instructions were then utilized to detect insulin using 3,3-diamonobenzidine (DAB) as the chromogenic substrate using light microscopy.