Encinas et al. 10.1073/pnas.0601992103. |
Supporting Figure 7
Supporting Materials and Methods
Fig. 7. Fluoxetine does not affect neurogenesis in the subventricular zone (SVZ). (A) SVZ of the lateral ventricle of the nestin-CFPnuc mouse, stained for CFPnuc (red). (B and C) Fluoxetine treatment, analyzed 1 day after BrdU administration, does not affect the number of BrdU-positive cells (B) or nestin-CFPnuc cells (C). V, vehicle; F, fluoxetine. (D and E) Representative photomicrographs of SVZ from control (injected with vehicle) (D) and fluoxetine-treated (E) animal after staining for CFPnuc (red) and BrdU (green). (Scale bars: A, 50 mm; D and E, 5 mm.)
Supporting Materials and Methods
Generation of Transgenic Mice.
Nestin-GFP plasmid (1) containing a promoter and the second intron of the nestin gene and polyadenylation sequences from simian virus 40 was used, after substituting GFP for cyan fluorescent protein with nuclear localization (CFPnuc) domain, to generate nestin-CFPnuc construct. The vector backbone was removed by SmaI digestion and purification, and the resulting 8.8-kb fragment was used for pronuclear injections into the fertilized oocytes from C57BL/6xBalb/cBy hybrid mice. Several independent lines were generated, and two lines were used for the subsequent analysis. They produced identical patterns of CFPnuc expression. Transgenic mice were repeatedly mated with C57BL/6 mice for >7 generations. Use of animals was reviewed and approved by the Cold Spring Harbor Laboratory Animal Use and Care Committee.Immunohistochemistry
. Mice were fixed by transcardial perfusion with 30 ml of 4% (wt/vol) paraformaldehyde in PBS, pH 7.4. The brains were removed, cut longitudinally into two hemispheres, and postfixed for 3 h at room temperature, then transferred to PBS and kept at 4°C. Serial sagittal sections, 50-mm thick, were cut with a Vibratome 1500 (Vibratome, St. Louis). Immunostaining was carried by following the standard procedure. The sections were incubated with the primary antibodies at 4°C in PBS containing 0.2% Triton X-100 and 3% BSA. After thorough washing with PBS, secondary antibodies were incubated in the same solution for 1 h at room temperature. After washing with PBS, the sections were mounted on gelatin coated slides with DakoCytomation Fluorescent Mounting Medium (Dakocytomation, Carpinteria, CA). For the analysis of BrdU incorporatrion, ssections were treated with 2 M HCl for 30 min at 55°C, washed with PBS, treated with 1 mM sodium tetraborate for 10 min at room temperature, and finally washed with PBS. The antibodies used are as follows: chicken anti-GFP (Aves Laboratories, Tigard, OR; 1:500); guinea pig anti-GFAP (Advanced Immunochemicals, Long Beach, CA; 1:500); mouse anti-nestin (Chemicon International, Temecula, CA 1:100); chicken anti-vimentin (Chemicon; 1:2,000); Sox2 (Chemicon; 1:500); rabbit anti-BFABP (gift from Carmen Birchmeier, Max Delbrück Center for Molecular Medicine, Berlin); mouse anti-NeuN (Chemicon; 1:800); rabbit anti-Dcx (gift from Dr. C. A. Walsh, Harvard Medical School, Boston; 1:2500); rabbit anti-Prox 1 (gift from Dr. S. J. Pleasure, University of California, San Francisco; 1:2,000); rat anti-BrdU (Serotec; 1:400); mouse anti-PSA-NCAM (Chemicon; 1:400); AlexaFluor 488 goat anti-chicken (Molecular Probes; 1:500); AlexaFluor 594 goat anti-mouse (Molecular Probes; 1:500); AlexaFluor 568 goat anti-rat (Molecular Probes; 1:500); Cy5 goat anti-guinea pig (Jackson Immunoresearch; 1:500); Cy5 goat anti-rabbit (Jackson Immunoresearch; 1:500); and Texas Red donkey anti-chicken (Jackson Immunoresearch; 1:500).Quantification.
Quantitative analysis of cell populations was performed by means of design-based (assumption free, unbiased) stereology (2). Slices were collected by using systematic-random sampling. One brain hemisphere was randomly selected per animal. The hemisphere was sliced sagittaly, in a lateral-to-medial direction, from the beginning until the end of the lateral ventricle, thus including the whole DG. The 50-mm slices, obtained with a 1500 Vibratome (Vibratome) were collected in six parallel sets, each set consisting of 12 slices, each slice 300 mm apart from the next.For counts in the DG, all of the cells of each type described were counted in every slice, excluding those in the uppermost focal plane. The volume of the reference space (the GCL plus the SGZ) was calculated by the Cavalieri point method. In the SVZ, for BrdU counts, all of the BrdU stained cells were counted in each slice, excluding those in the uppermost focal plane. For nestin-CFP cells, the two-stage approach was used. At least 10 optical disectors (20 × 20 × 20 mm), placed by following a systematic-random manner, were applied on each slice. Then the volume of the space reference (the SVZ, defined by the presence of nestin-CFP+ cells) was estimated by using the Cavalieri point method.
1. Mignone, J. L., Kukekov, V., Chiang, A. S., Steindler, D. & Enikolopov, G. (2004) J. Comp. Neurol. 469, 311–324.
2. Peterson, D. A. (1999) Methods 18, 493–507.