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Cell cycle analysis in the rat external granular layer evaluated by several bromodeoxyuridine immunoperoxidase staining protocols

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Abstract

An important step in bromodeoxyuridine (BrdU) immunohistochemistry is the production of single-stranded DNA to make the incorporated BrdU accessible to the antibodies. This paper examines the effect of distinct DNA denaturation pretreatments (DNase I, sodium citrate buffer, endonuclease Eco RI and exonuclease III, and HCl hydrolysis) on detection of BrdU. We found that all the methods used in the partial denaturation of DNA combined good nuclear immunostaining with acceptable tissue integrity. We also observed that these immunohistochemical protocols revealed a spatial pattern in the distribution of DNA-synthesizing cells within the cerebellar external granular layer (EGL) of 10-day-old rats, allowing us to estimate the fraction of S-phase cells. Our results indicate that detection of BrdU-stained cells is affected by the distinct histological procedures used in such detection. Additionally, as the duration and phases of the cell cycle in EGL neuroblasts are estimated in accordance with BrdU detection, an effect on this detection can render the measurement of cell cycle inaccurate. The present work shows that DNase I and citrate buffer, at appropriate conditions, may be good alternatives for acid denaturation. However, they are less sensitive than autoradiographic techniques that use 3H-thymidine administration. Finally, current data reveal that short survival times after a single BrdU exposure do not seem to affect the cell cycle progression of the EGL neuroblasts.

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Acknowledgements

The authors are grateful to Dr María del Carmen Santa-Cruz and Dr José Pablo Hervás for providing the rats used in the experiments described in this discussion.

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Authors and Affiliations

  1. Unidad de Citología e Histología, Facultad de Biociencias, Universidad Autónoma de Barcelona, Bellaterra, 08193, Barcelona, Spain

    Vanesa Molina, Lucía Rodríguez-Vázquez & Joaquín Martí

  2. Departament de Filologia Anglesa i de Germanística, Àrea de Filologia Anglesa, Bellaterra, 08193, Barcelona, Spain

    David Owen

  3. Servei d’Estadística Aplicada, Universidad Autónoma de Barcelona, Bellaterra, 08193, Barcelona, Spain

    Oliver Valero

Authors
  1. Vanesa Molina
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  2. Lucía Rodríguez-Vázquez
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  3. David Owen
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  4. Oliver Valero
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  5. Joaquín Martí
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Correspondence to Joaquín Martí.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Informed consent was obtained from all individual participants included in the study.

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Molina, V., Rodríguez-Vázquez, L., Owen, D. et al. Cell cycle analysis in the rat external granular layer evaluated by several bromodeoxyuridine immunoperoxidase staining protocols. Histochem Cell Biol 148, 477–488 (2017). https://doi.org/10.1007/s00418-017-1593-1

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  • DOI: https://doi.org/10.1007/s00418-017-1593-1

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