Data are expressed as the total number of BrdU-positive cells ± SEM. The same investigator performed all the quantification of the RMS and SGZ to reduce inter-observer variation in cell counting parameters. Also, the identity of the mice from which the sections were generated was unknown to the investigator during the data collection phase. We used the cumulative BrdU labeling protocol to measure and compare the lengths of the cell cycle and S phase of the rapidly dividing cell populations in the RMS of C57BL/6J and A/J mice (Nowakowski et al.,
1989). Administration of BrdU and tissue preparation were as described above. Consecutive sections were cut at 8-μm thickness, stained with anti-BrdU and counterstained with CV. Using a 40× objective, we determined the labeling index (LIt) – the ratio
of BrdU-positive cells to the total RMS cell population at a given time (t) – in brains obtained from animals Compound Library manufacturer killed at t = 0.5, 2.5, 4.5, 6.5, 8.5 and 10.5 h after the first BrdU injection. As the RMS is a long, compact cellular architecture, we estimated the total cell population by selecting four representative segments along the course of each RMS (two from the vertical arm, one from the RMS elbow and one from the horizontal arm depicted in supplementary Fig. S2), Venetoclax mw counted all cells within these segments and measured the corresponding area (mean value of each segment is 4500 μm2) to obtain the estimated cell density of the RMS. RMS lengths and areas were measured using AnalySIS Opti Version 3.3.776 software (Soft Image System). The density was then multiplied by the total RMS area to estimate the total cells in an RMS. CHIR-99021 Once the LIs at every time point were calculated for
each genotype, the average LI (y-axis) was plotted against the time after the first BrdU injection (x-axis). We used the equation, LI0 = GF × Ts/Tc, to calculate the length of the S phase (Ts) and the length of the cell cycle (Tc) (Nowakowski et al., 1989) where LI0 is the labeling index at the time of the first BrdU administration (t = 0) and is equivalent to the y-intercept of the graph. Growth fraction (GF) is the proliferating proportion of the total RMS population and it is equivalent to the maximum LI plotted in the graph where all proliferating cells in the RMS are assumed to be labeled by BrdU at least once (GF = LIt; t ≥ Tc − Ts). Ts and Tc were subsequently calculated using a non-linear least squares fit to the labeling index curve (Nowakowski et al., 1989). Three mice from each genotype used in the cell cycle analysis were also used for a full reconstruction and quantitative analysis of the RMS to obtain the total volume and total number of cells in the RMS of each genotype. We used NeuroLucida and Neuroexplorer software (version 4, 2000 by MicroBrightField, Inc.).