2010) asymmetric grooming, the rats display an uninterrupted series of at least three wash strokes directed to the stimulated area. The score was assumed to reflect the magnitude of the aversiveness
evoked by the mechanical stimulation, being equal to zero in the case of absence of response. A mean score value was then calculated for each stimulation series. Immunohistochemical analysis The rats were deeply anesthetized with urethane (1.5 g/kg i.p.). Twenty minutes after the induction of anesthesia, rats were stimulated for 2 min on one infraorbital region by gentle air puffing (60 stimuli delivered, 0.5 Hz). Three minutes after the end of stimulation, the rats were perfused transcardially with warm (37°C) heparinized saline (25 Inhibitors,research,lifescience,medical IU heparin/mL) followed Inhibitors,research,lifescience,medical by cold (10°C) phosphate-buffered solution (0.1 mol/L, pH 7.6) containing 4% paraformaldehyde and 0.03% picric acid for 15 min. The brainstem was then removed and transferred to the same fixative solution for 1 h and then placed in 30% sucrose and 0.05% sodium azide solution overnight at 4°C. Coronal 40-μm thick sections of the brainstem
were cut on a freezing microtome (Leica, Wetzlar, Germany) and collected in a 0.05 mol/L Tris-buffered saline (TBS). For immunofluorescence, free-floating Inhibitors,research,lifescience,medical brainstem sections were placed in 1% normal goat serum for 1 h before overnight incubation at room temperature in primary antibody solutions (mouse and rabbit antiphosphorylated extracellular signal-regulated kinases 1/2 [pERK1/2] [1:1000, Cell Signaling Technologies, Danvers, MA], and mouse and rabbit anti-PKCγ [1:4000, Sigma-Aldrich and Santa Cruz, Dallas, TX]). The corresponding secondary antibodies (1:400 for goat anti-mouse Cy3, 1:200 for goat anti-rabbit Cy2) were incubated Inhibitors,research,lifescience,medical at room temperature for 3 h. All antibodies were diluted in TBS containing 0.25% bovine serum albumin and 0.3% Triton X-100. The sections were finally rinsed in TBS, mounted on gelatin-coated slides, dehydrated in alcohol, scientific study cleared in xylene, and coverslipped Inhibitors,research,lifescience,medical with 1,3-diethyl-8-phenylxanthine (DPX) medium. The specificity of the immunostaining was assessed
by omitting the primary antibodies, which resulted in the absence of signal. Immunofluorescent staining was analyzed by using a motorized Brefeldin_A Zeiss R115777 Axioplan 2 microscope equipped with a Hamamatsu C4742-95 digital camera (Hamamatsu Photonics France SARL, Massy, France) (switching between FITC and Texas Red filter sets) driven by MetaMorph® 5.4 software. In each rat, image acquisition and fluorescent signal quantification were done using seven different sections, each taken at a given rostrocaudal plane within the MDH (from 0 to −2160 μm at 360 μm intervals). To reduce the variability in staining between sections, the same area of the MDH was taken in consideration for quantification of the fluorescent signal. Measurement of PKCγ fluorescence was taken from the layer IIi as it contained the majority of interneurons positive for this marker.