Microfire digital camera

As previously reported, DA neurons and macrophages of the myenteric plexus were identified on free-floating sections following immunohistochemistry (Côté et al., 2011 (link)). Briefly, myenteric ganglia neurons were stained with Cuprolinic blue for 60 min at 37°C (Holst and Powley, 1995 (link)). DA neurons and macrophages were identified with a polyclonal tyrosine hydroxylase antibody (TH; 1:1000; Cedarlane, ON, Canada) and the pan-macrophage/microglia marker ionized calcium-binding adaptor molecule 1 (Iba-1; 1:1000; Cedarlane), respectively. Biotinylated goat anti-rabbit IgG (1:1000; Cedarlane) was used as a secondary antibody. The signal was revealed with the ABC Vectastain Elite kit (Vector Laboratories, Inc., ON, Canada) and 3,3′-diaminobenzidine (DAB, Vector Laboratories, Inc.) as chromagen. All pictures were acquired with a Nikon C80i microscope equipped with a MicroFire digital camera (MBF Bioscience, Williston, VT). Figures were assembled using Adobe Illustrator CS3.

Stereological analyses were performed with the Stereo Investigator software 6.0 (Microbrightfield, VT, United States) operated by an observer (blind to treatments), as previously described (Côté et al., 2011 (link)). The contours of the whole mounted sections were traced as a virtual overlay over images through a ×4 objective lens in brightfield mode (Nikon C80i microscope equipped with a MicroFire digital camera; MBF Bioscience, Williston, VT). TH⁺ and Iba-1⁺ cell bodies were counted only if they did not cross the forbidden contours. Densities were calculated as the number of cells divided by the traced area (in mm²). MHCII and VIP quantifications were performed using the NIH ImageJ software (Wayne Rasband, Bethesda, MD, United States). Section contours were also traced as a virtual overlay on images with the freehand selection option. VIP immunofluorescent staining quantification was executed on gray scale values images and measured by the mean pixel intensity. MHCII⁺ cell count was performed using the embedded cell counter plugin and densities were calculated as the number of cells per area unit (in mm²). All figures were assembled using Adobe Illustrator CS3.