2.2.1 Triple-label immunocytochemistry:
25 male Wistar rats were killed by stunning and cervical dislocation under Schedule 1 of the United Kingdom Animals (Scientific Procedures) Act 1986. Then lumbar (L4-L6) DRG were excised under aseptic conditions and axons were trimmed away using micro-scissors. DRG were immersed in Tissue-Tek (Thermo Scientific, Raymond Lamb) and rapidly frozen in a hexane bath cooled with dry ice (-73°C ) for 20 min in a Dewar flask and then transferred for storage at -20°C until used. Tissue blocks were trimmed and 10μm sections of DRG were cut using an ultracryomicrotome (model OTF, Hacker-Bright OTF, Fairfield, NJ), and thawed onto poly-L-lysine coated glass slides (VWR International, Lutterworth, UK) and allowed to air dry.
Sections were fixed with 2% paraformaldehyde in 0.1 M phosphate buffer for 10min at ambient temperature, followed by a wash in PBS for 15 min. For the dilution of both primary and secondary antibodies, PBS with 10% of host- directed serum was used.
The sections were incubated in a blocking buffer for 30 min at room temperature in an incubation box. Triton X-100 is a detergent and facilitates the penetration of the antibody into the tissue sections and the cells. Sections were washed in PBS (2 x 15 min) and were incubated in the appropriate primary antibody overnight in an incubation box at 4°C (the primary antibodies for EP4 (goat anti-rabbit, 1:200), TRPV1 (goat anti-mouse; 1:1000) and B2 (goat anti-donkey; 1:200))
Control experiments were performed to determine the level of nonspecific binding and to achieve this primary antibody was omitted from the incubation medium. The sections were then washed in PBS (6 x 10 min) after which they were placed in a solution containing the appropriate secondary antibody donkey anti-rabbit (for EP4) DyLight 405 conjugated secondary antibody (1:1000; Jackson ImmunoResearch Labs and a donkey anti-goat (for B2) TR- conjugated secondary antibody (1:400; Jackson ImmunoResearch Labs) and a donkey anti-mouse (for TRPV1) AF-conjugated secondary antibody (1:500; Jackson ImmunoResearch Labs). and incubated for 2 hours at room temperature. Sections were then washed in PBS (2x15 min) and tissue sections were mounted using a glycerol-based, aqueous, antifade mountant, Citiflour (UKC Chem. Laboratory, Kent, UK).
The mounted sections were photographed using a Nikon Labphot 2A epifluorescence microscope using the appropriate filter set (FITC-absorbance peak = 492nm, emission peak = 520nm; TR-absorbance = 596nm, emission peak= 620nm) for each fluorophore with a x20 objective lens. Publication-quality micrographs were captured using laser-scanning microscope confocal microscope (Olympus FluoView® 300), employing an Argon ion (488nm) and green HeNe (543nm) lasers.