The next day, sections were rinsed with 0.1 m PBST, incubated in biotinylated horse anti-mouse IgG (1 : 200, Vector Laboratories, Burlingame, CA, USA) for1 h, and rinsed again with 0.1 m PBST. Tissue sections were then treated with solutions from the VECTASTAIN Elite ABC kit (Vector Laboratories) according to the supplier’s instructions for 30 min at room temperature followed by washes in 0.1 and 0.001 m PB. Immunoreactivity was detected using 3, 3′-diaminobenzidine (DAB; Sigma-Aldrich) at 25 mg/50mL in 0.1 m PB with 0.004% H2O2. Sections were thoroughly c-Met inhibitor rinsed with dH2O, dehydrated and then coverslipped. To determine the
number of BrdU-positive cells in the RMS, we first located the RMS by staining every tenth section throughout the left hemisphere with anti-BrdU, and then identified the single sagittal section within the 10-series that had the greatest representation of the RMS for analysis. The distribution of 1-h-labeled BrdU cells was highly localized in the RMS, which begins at the rostral tip of the lateral ventricle and terminates at the caudal end of the olfactory bulb (Fig. 1). The linear density of BrdU-positive cells per millimeter of RMS length was calculated from a single section that contained the most intact RMS exhibiting the stereotypical trajectory of proliferating cells en CP-868596 chemical structure route to the OB. BrdU-immunoreactive
cells in the RMS of this optimal section were counted under brightfield illumination and with the aid of a 20× objective (Zeiss 200M Axiovert inverted microscope equipped with Axiovision 4.6 software). RMS length was measured using NIH ImageJ (version 1.42) software. Linear density from 1 h BrdU labeling
was systematically determined for A/J, C57BL/6J and their RI strains and was expressed as mean ± SEM for each strain. Another counting approach adapted from Lee et al. (2003) was used in which we counted the number of BrdU-positive cells in every tenth immunostained section (80-μm intervals) throughout the entire medial to lateral extent of the RMS. The total number of labeled cells was calculated for 20 randomly selected animals and this value is highly Glycogen branching enzyme correlated with the linear density (R = 0.88; P < 0.0001; see Supplementary material Fig. S1), thus demonstrating the effectiveness of our single best-section quantification method. Animals used for analysis of BrdU-labeling in the RMS were also used to examine the proliferative activities in another neurogenic site, the subgranular zone (SGZ) of the hippocampal dentate gyrus. We quantified BrdU-positive cells in the SGZ, which is located at the interface between hilus and the granular layer of the dentate gyrus (DG), and this proliferative layer can be easily visualized by cresyl violet (CV) stain under a 40× objective (Kempermann et al., 2003).