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. 2009 Nov 10;517(2):123-33.
doi: 10.1002/cne.22107.

Thymidine analog methods for studies of adult neurogenesis are not equally sensitive

Benedetta Leuner  1 , Erica R Glasper, Elizabeth Gould
Affiliations

Thymidine analog methods for studies of adult neurogenesis are not equally sensitive

Benedetta Leuner et al. J Comp Neurol. .

Abstract

Adult neurogenesis is often studied by labeling new cells with the thymidine analog bromodeoxyuridine (BrdU) and using immunohistochemical methods for their visualization. Using this approach, considerable variability has been reported in the number of new cells produced in the dentate gyrus of adult rodents. We examined whether immunohistochemical methods, including BrdU antibodies from different vendors (Vector, BD, Roche, Dako, Novocastra, and Accurate) and DNA denaturation pretreatments alter the quantitative and qualitative patterns of BrdU labeling. We also compared the sensitivity and specificity of BrdU with two other thymidine analogs, iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU). We found that the number of BrdU-labeled cells in the dentate gyrus of adult rats was dependent on the BrdU antibody used but was unrelated to differences in antibody penetration. Even at a higher concentration, some antibodies (Vector and Novocastra) stained fewer cells. A sensitive BrdU antibody (BD) was specific for dividing cells; all BrdU-labeled cells stained for Ki67, an endogenous marker of cell proliferation. We also observed that DNA denaturation pretreatments affected the number of BrdU-labeled cells and staining intensity for a marker of neuronal differentiation, NeuN. Finally, we found that IdU and CldU, when used at molarities comparable to those that label the maximal number of cells with BrdU, are less sensitive. These data suggest that antibody and thymidine analog selection, as well as the staining procedure employed, can affect the number of newly generated neurons detected in the adult brain, thus providing a potential explanation for some of the variability in the adult neurogenesis literature.

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Figures

Figure 1
Figure 1
BrdU antibodies do not label the same number of cells in the dentate gyrus. (A) BD and Roche antibodies detected significantly more BrdU-labeled cells in the dentate gyrus with a 2h post-BrdU survival time compared to Vector. The number of BrdU-labeled cells did not differ between BD and Roche. (B) In a second comparison, we extended our analysis to include three other BrdU antibodies (Dako, Novocastra, Accurate) in addition to BD. Two hours following BrdU administration, Novocastra detected the fewest number of BrdU-labeled cells compared to all other antibodies, while the remaining antibodies did not differ from each other. (C) Similar results were obtained 3 weeks following BrdU administration. Vector and Novocastra detected the fewest number of BrdU-labeled cells relative to all other antibodies examined, with the remaining antibodies not differing significantly from each other. Bars represent mean ± SEM, * P < 0.05.
Figure 2
Figure 2
BrdU antibodies, analogs, and pretreatments produce variable histology. (A, B) BrdU-labeled cells (arrows) in the dentate gyrus from sections stained with Vector (A) or BD (B) antibodies 3 weeks after BrdU administration. High magnification examples of BrdU-labeled cells in the granule cell layer (gcl) 2h (C, D) and 3 weeks (F, G) after BrdU administration as detected with Vector (C, F) or BD (D, G). At both the 2h and 3 week timepoints, more BrdU-labeled cells were detected with the BD antibody. (E, H) High magnification examples of IdU- and CldU-labeled cells in the gcl 2h after equimolar delivery of the analogs as detected with the BD antibody. IdU and CldU labeled fewer cells than BrdU and could not be discriminated because of antibody cross-reactivity. (I-K) Immunofluorescence double-labeling of BrdU (I) and Ki67 (J). Merged image shown in (K). All BrdU-labeled cells detected with the BD antibody stained for Ki67 indicating specificity for dividing cells. (L–P) Confocal images of NeuN staining from sections pretreated with HCl alone (L), HCl + formamide (M, N) or Steam heating (O, P). HCl + formamide and Steam heating pretreatments were variable in their staining patterns, some had high background (M, O) whereas others had no nuclear staining (N, P). Scale bars, 10 µm.
Figure 3
Figure 3
Less sensitive BrdU antibodies are not made more sensitive by increasing their concentration. At both the 2h (A) and 3 week (B) survival times, a 1:100 concentration of the Vector antibody labeled fewer cells in the dentate gyrus compared to the Dako antibody. In contrast, a 1:100 concentration of the Novocastra BrdU antibody amplified the number of BrdU-labeled cells at both 2h and 3 weeks. However, at 3 weeks, the number of cells labeled by Novocastra was lower than that labeled by Dako. Bars represent mean ± SEM, * P < 0.05 vs. Dako, ** P < 0.05 vs. Dako and Vector.
Figure 4
Figure 4
Denaturation pretreatment methods affect the number of BrdU-labeled cells and staining for the mature neuronal marker, NeuN, in the dentate gyrus. (A) Fewer BrdU-labeled cells were detected with Steam heating on tissue stained using peroxidase methods. (B) Tissue stained for NeuN immunofluorescence using the HCl alone pretreatment had a substantially higher signal:noise ratio than tissue stained for NeuN using either HCl + formamide or Steam heating pretreatments. Bars represent mean ± SEM, * P < 0.05.
Figure 5
Figure 5
The thymidine analogs, IdU and CldU, are not as sensitive as BrdU. Overall, BrdU-labeled cells in the dentate gyrus were more abundant than either IdU- or CldU-labeled cells (** P < 0.05) suggesting that these thymidine analogs are not labeling cells undergoing S-phase with equal probability. Also, BrdU antibodies did not label IdU or CldU with specificity. Although BD detected more IdU-labeled cells than Accurate, some IdU-positive cells were found with the Accurate antibody. In addition, both BD and Accurate detected a substantial number of CldU-labeled cells. Bars represent mean ± SEM, * P < 0.05.
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