2012) as specified by the manufacturer (see the technical inform

2012) as specified by the manufacturer (see the technical information data sheet). Cytochrome oxidase (CO) staining Two rats were used to verify the possibility of using the technique in this series of experiments. Animals were perfused and tissues postfixed and cut as described

above. Brains were freeze sectioned in the sagittal plane. Two consecutive sections, one per stereotaxic plane, were used in two different ways. The first sections (60 μm thick) were incubated at 37°C in the dark for 10–12 Inhibitors,research,lifescience,medical h in a solution containing 50 mg DAB, 30 mg cytochrome C (Type III, Sigma), and 4 g sucrose dissolved in 90 mL PB (0.1 mmol/L, pH 7.4; Wong-Riley 1979). Incubation was arrested when a clear differentiation between cerebral cortex and cc was visible. Sections were rinsed many times in PB, mounted on subbed slides, air-dried, dehydrated Inhibitors,research,lifescience,medical in xylene and then

coverslipped. The adjacent sections (40 μm thick) were counterstained with neutral red (1% in aqueous solution) and then coverslipped. Selected sections from CC-NADPH-1/11, CC-nNOS-1/6 and CC-Fl-1 and -2 were used for CO staining as described above. Data analysis The distribution of NADPH-d+ Inhibitors,research,lifescience,medical and NOSIP neurons in the cc was obtained using a camera lucida attached to a Leitz Orthoplan microscope equipped with a 25× objective. Callosal boundaries were obtained by comparing the sections reacted for COHi with those counterstained with neutral red. The reconstructions thus obtained were

then compared with those of the atlas of Paxinos Inhibitors,research,lifescience,medical and Watson (1982). Counts were performed in serial sagittal sections from two animals (CC-NADPH-10-11; see Table ​Table2).2). The total number of intracallosal NADPH-d+ neurons was calculated on sections from both hemispheres starting from the lateral 4.5 sagittal plane of one hemisphere to reach Inhibitors,research,lifescience,medical the same stereotaxic plane in the contralateral hemisphere. Table 2 (A) Arbitrary subdivisions of the rat cc, (B) Number of NADPH-d+ neurons in the three subdivisions of the rat cc Microscopic studies of the morphology and percentage of intracallosal neurons (three cases: CC-NADPH-d-5; -7; -9; see Tables ​Tables3,3, ​,4)4) were Batimastat performed using staining criteria similar to those of previous Golgi and NADPH-d studies (Jacobs and Scheibel 1993; Jacobs et al. 1993; Phillips et al. 2000; Barrera et al. 2001). Selected neurons, drawn using a camera lucida and a 100× oil immersion objective, exhibited the following characteristics: (1) labeled neurons had a clearly selleck chemicals Gefitinib distinguishable morphology; (2) cell bodies had a central location within the 60-μm section depth to minimize the cutting of dendritic branches near the section surface; (3) dendrites were not overly obscured by other heavily stained processes from nearby cells; (4) dendritic trees were intensely labeled and did not show discontinuity with their cell bodies.

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