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. 2007 May 30;27(22):5837–5844. doi: 10.1523/JNEUROSCI.5048-06.2007

Figure 1.

Figure 1.

Intensity of BrdU, Pax6, and DNA label is stronger in tissue pretreated by the steamer/citrate method compared with tissue pretreated with HCl. A, Parasagittal cryostat section of postnatal day 1 (P1) rat forebrain labeled with Hoechst 33342, which labels all cell nuclei. Images in the figures of this study show photomicrographs that include the SVZa as well as part of the adjacent RMS, lateral ventricle (LV), and overlying corpus callosum (for example, to the right of the dashed line in B–F). In Hoechst-labeled sections, the SVZa and RMS, regions of high cell density and therefore high nuclear density, appear quite bright compared with the bordering tissue, which has a much lower density of cells. In the neonate, neuronal progenitors migrate from the SVZa along the RMS to the olfactory bulb, in which they become interneurons. The cells in the SVZa/RMS are highly proliferative, which makes them useful regions for comparing BrdU-labeling techniques. This montage was assembled from images of the same steamer/citrate-pretreated section used for B; the box outlines the field shown in B. B1–D4, Parasagittal sections of P1 rat brain on glass slides that were pretreated by being covered with 10 mm sodium citrate buffer, pH 6.0, and heated in a steamer for 15 min (B1–B4), pretreated by immersion in 2.0 m HCl at 40°C for 20 min (C1–C4), or not pretreated (D1–D4). After pretreatment, all sections were processed and labeled in parallel using the same antibody solutions and procedures. BrdU is incorporated into replicating DNA, Pax6 is a transcription factor protein, and the nuclear dye Hoechst 33342 binds to double-stranded DNA. All three labels are expected to have a principally nuclear distribution. The three sections shown (B–D) are adjacent sections from a single rat brain; these sections have similar histological features and would therefore be expected to exhibit a similar pattern of labeling. For each type of pretreatment tested, a field dominated by the SVZa was sequentially imaged for BrdU (B1, C1, D1; 4 s exposure time), Pax6 (B2, C2, D2; 6 s exposure), and Hoechst (B3, C3, D3; 180 ms exposure). In the merged images (B4, C4, D4), BrdU label is red and Pax6 is green. Solid lines demarcate the border of the lateral ventricle, and dashed lines outline the boundary between the SVZa and adjacent neural tissue. Within each row, the solid and dashed lines are identically positioned in each image. It is noteworthy that the nonspecific rhodamine channel fluorescence was lower for sections pretreated with the steamer/citrate method than for sections pretreated with HCl. BrdU labeling is much brighter for the section that received steamer/citrate pretreatment than for the section pretreated with HCl (compare B1 with C1); no BrdU labeling is detected in the section that received no pretreatment. Pax6 labeling of the steamer/citrate-pretreated section (B2) is much brighter than for the HCl-pretreated (C2) and no pretreatment (D2) sections. Hoechst labeling is appreciably stronger for the steamer/citrate pretreatment (B3) than for no pretreatment (D3), and Hoechst labeling is absent from the HCl-pretreated section (C3). E, F, BrdU label of steamer/citrate-pretreated sections imaged with a shorter exposure time (2 s; E) than in B (4 s) or incubated with more dilute antibody (F). The image in E is the same field as in B1–B4; the image in F is from an adjacent section labeled using the BrdU antibody at a 1:2000 dilution (4-fold more dilute than in B). Note that despite incubation with more dilute antibody and a shorter exposure time, the BrdU label in the steamer/citrate-pretreated section (F) is still brighter than in the HCl-pretreated section (C1). As in B–D, solid lines demarcate the border with the lateral ventricle; dashed lines outline the boundary between the SVZa and adjacent neural tissue. G, Quantitative comparison of density of cells scored as BrdU(+) in steamer/citrate versus HCl-pretreated sections. The density of BrdU(+) cells in the SVZa of three animals was quantified in adjacent sections pretreated either with the steamer/citrate method or HCl. The density of cells scored BrdU(+) is similar, although optimal exposure time for imaging BrdU label in the HCl-pretreated section required an exposure three times longer than that for imaging the steamer/citrate-pretreated slide (3 vs 1 s at 40×). A, Anterior; D, dorsal; LV, lateral ventricle. Scale bars: A, 500 μm; D4 (for B1–F), 200 μm.