*4.6. Immunohistochemistry*

According to Terayama et al. [116] and Panneton et al. [117], the central afferent innervations of the IoN are mostly distributed in (but not restricted to) the dorsal and lateral part of the Sp5C, projecting to all the laminae. The pattern of CGRP and SP protein related to the painful component of the IoN was investigated in the superficial laminae of the Sp5C.

Immediately after the MST test, animals were anaesthetized and perfused transcardially with phosphate buffered saline (PBS) and 4% paraformaldehyde. The medullary segment containing the Sp5C, between +1 and −5 mm from the obex, was removed and post-fixed for 24 h in the same fixative; subsequently, samples were transferred in solutions of sucrose at increasing concentrations (up to 30%) during the following 72 h. All samples were cut transversely at 30 μm on a freezing sliding microtome. CGRP and SP protein expression was evaluated using the free-floating immunohistochemical technique, as previously reported [24]. For CGRP we used an anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:3200, and an anti-rabbit antibody (Chemicon, Temecula, CA, USA) at a dilution of 1:5000 for SP; both primary antibodies were incubated for 24 h at room temperature. After incubation at room temperature with the secondary biotinylated antibody (Vector Laboratories, Burlingame, CA, USA) and the avidin-biotin complex (Vectastain, Vector Laboratories), sections were stained with the peroxidase substrate kit DAB (3 3 -diaminobenzidine tetrahydrochloride) (Vector Laboratories, Burlingame, CA, USA).

The area covered by CGRP and SP immunoreactive fibers in the Sp5C ipsilateral and contralateral to the surgery (12 sections per animal), was expressed as optical density (OD) values [24,114,118], acquired using an AxioSkop 2 microscope (Zeiss) and a computerized image analysis system (AxioCam, Zeiss, Göttingen, Germany) equipped with dedicated software (AxioVision Rel 4.2, Zeiss, Göttingen, Germany). All sections were averaged and reported as the mean + SEM of OD values.
