Anti rabbit 546

The materials used included hexamethyldisiloxane (HMDS, Sigma-Aldrich, St. Louis, MO, USA); sodium fluoride (NaF, Sigma Chemical, St. Louis, MO, USA); trypsin (Trypsin Gold Mass Spectrometry, Promega, Madison, USA); PlusOne 2D Cleanup kit (GE Healthcare, Uppsala, Sweden) and 3 kDa AMICON (Millipore, St Charles, MO, USA). Antibodies: nNOS (H-299, sc-8309 Santa Cruz, Dallas, TX, USA), Mouse anti-HuC/D (A-21271, Invitrogen, Waltham, MA, USA), Rabbit anti-CGRP (AB15360, Millipore, St Charles, MO, USA), Rabbit anti-VIP (V0390, Sigma-Aldrich, St. Louis, MO, USA), Goat anti-substance P (sc9758, Santa Cruz, Dallas, TX, USA), Anti-rabbit 546 (A10040, Invitrogen, Waltham, MA, USA), Anti-mouse 488 (A21202, Invitrogen, Waltham, MA, USA), Anti-rabbit 546 (A10040, Invitrogen, Waltham, MA, USA), and Anti-goat 568 (A11057, Invitrogen, Waltham, MAs, USA).

For immunofluorescence, paraformaldehyde-fixed cells samples were permeabilized in 0.1% Triton X100 in PBS and incubated with blocking solution (10% normal goat serum, NGS, in 0.1% Triton X100 in PBS) for 1 h at room temperature [[36] (link), [37] (link), [38] (link)]. Samples were incubated overnight at 4 °C with the following primary antibodies diluted in PBS Rabbit anti-NOS2 (Santa Cruz, Cat#sc-7271; 1:100), mouse anti-ARG1 (Santa Cruz, Cat#sc-20150; 1:100). The following day, after washing, samples were incubated for 1 h at room temperature with the appropriate fluorescence goat secondary antibodies: anti-rabbit 546 (Invitrogen, Cat# A11010, RRID: AB_143156, 1:1000) and anti-chicken 488 (Abcam, Cat# ab150169, RRID: AB_2636803, 1:1000). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (Dapi, 1:1000, Cat# D1306, Invitrogen) for 5 min at room temperature. Slides were mounted with fluorescent mounting medium Permafluor (ThermoScientific) and digital images were acquired using a Leica DM IRB (Leica Microsystems, Buccinasco, Milano, Italy) fluorescence microscope or the Leica TCS SP8 confocal microscope.

Immunohistochemistry was performed on 4 μm paraformaldehyde fixed sections. Following rehydration, heat-induced antigen retrieval was performed with 10 mM sodium citrate buffer (pH 6) and sections were blocked in 15% goat serum before incubation with primary antibodies: chicken anti-gfp (Abcam), mouse anti-smooth muscle actin (Dako), and rabbit anti-von Willebrand factor (Dako). Fluorescent secondary antibodies were used for detection, goat anti-chicken 488, anti-rabbit 546, and anti-mouse 647 (Invitrogen), and slides were mounted with ProLong Gold Antifade Reagent with DAPI (Invitrogen). Images were acquired using a Zeiss LSM510 confocal imaging system (Carl Zeiss).