Bx41 wi microscope

The mesenteric arterial bed was perfused with Bouin’s solution to fix the mesenteric arteries. Second order arteries were isolated, permeabilized with 0.01% Triton X-100 and blocked with 3% BSA, as described previously^{67 (link)}. Vessels were incubated overnight at 4 °C with an anti-CGRP primary antibody (Thermo Scientific, Rockford, IL, USA), and then, with an Alexa-568-labeled goat anti-mouse secondary antibody (Molecular Probes, Eugene, OR, USA) for 4 h at 4 °C. The fluorescent signal was examined with an Olympus BX41 WI microscope and a CCD camera (Jenoptik ProgRes C5).

Astrocyte monolayers were fixed with 2% paraformaldehyde in PBS, blocked with 0.5% BSA in PBS and incubated with the corresponding rabbit polyclonal primary antibody, mouse monoclonal primary antibody or both in the case of co-immunofluorescence analysis, and then, with the appropriate Alexa-568-labeled goat anti-rabbit or anti-mouse secondary antibody (Invitrogen Molecular Probes, USA, Cat. #A11011 or Cat. #A11004, respectively), or Alexa-488-labeled goat anti-rabbit or anti-mouse secondary antibody (Invitrogen Molecular Probes, USA, Cat. #A11008 or Cat. #A10680, respectively) using the Signal Enhancer HIKARI (Nacalai Tesque, INC, Japan) as indicated by the manufacturer. The fluorescent signal was examined using an Olympus BX41 WI microscope and a CCD camera (Jenoptik ProgRes C5).

Cells were fixed with 1% formaldehyde and then washed with PBS solution (composition in mM: 136.9 NaCl; 2.68 KCl; 10.44 NaH₂PO₄; 1.76 KH₂PO₄) adjusted to pH 7.4. Nonspecific protein binding sites were blocked with PBS containing 0.1% FBS. Cells were incubated overnight with an anti-MYC monoclonal antibody to detect 5-HT₃A receptor (1:250, Thermo Fisher Scientific, Cat. # PA1-981, Rockford, IL, USA) and an anti-HA monoclonal antibody to label P2X4 receptor (1:250 Thermo Fisher Scientific, Cat. # 26183, Rockford, IL, USA). Cells were washed three times for 10 min with PBS and then incubated for 1 h with a secondary antibody conjugated to Alexa Fluor^®555 for anti-MYC (1:500, Cat. #A-21424, Molecular Probes, Eugene, OR, USA) or Alexa Fluor^®488 for anti-HA (1:500, Cat. # A-11029, Molecular Probes, Eugene, OR, USA). Then, cells were washed and mounted with Fluoromount-G (Electron Microscopy Sciences, Cat. # 17984-25, Hatfield, PA, USA). The fluorescent signal was examined using either an Olympus IX81 confocal inverted microscope coupled with an ORCA R2 Hamamatsu CCD camera or an Olympus BX41 WI microscope coupled with a Jenoptik ProgRes C5 CCD camera. As a negative control, primary antibodies were omitted.