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DNAse I pre-treatment markedly enhances detection of nuclear cyclin-dependent kinase inhibitor p57Kip2 and BrdU double immunostaining in embryonic rat brain

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Abstract

As a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs), p57Kip2 binds tightly to G1 cyclin/cyclin-dependent kinase complexes to block cell cycle progression. CKIs play critical roles in regulating the transition from proliferation to differentiation in many tissues, including the nervous system. Conversely, CKI dys-regulation contributes to neoplasia and cancer progression. While the combined detection of CKI immunoreactivity and S phase entry using bromodeoxyuridine (BrdU) incorporation may be particularly informative, successful immunostaining may be limited due to “masked” antigen epitopes and acid-induced signal degradation. We now report an improved double immunofluorescent method for detecting p57Kip2 and BrdU in paraformaldehyde-fixed frozen sections of embryonic rat brain. We substituted deoxyribonuclease I (DNAse I) for HCl pre-treatment to expose antigenic sites in frozen sections, and employed a biotinylated tyramide-based system to enhance p57Kip2 visualization. We identified a time- and dose-dependent relationship between DNAse I treatment and double labeling of p57Kip2 and BrdU, increasing both the numbers and intensities of immunopositive nuclei. With excess DNAse I treatment, however, there was signal degradation for both BrdU and total DNA, as reflected by DAPI staining. The use of DNAse I pre-treatment significantly increases the reliability and sensitivity of immunodetection of CKI nuclear factors, and should be useful for both developmental neurobiology studies as well as cancer diagnostic applications.

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Acknowledgment

This work was supported by NIH: grant R01 NS32401.

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  1. Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, RWJ SPH 362, Piscataway, NJ, 08854, USA

    Weizhen Ye, Georges Mairet-Coello & Emanuel DiCicco-Bloom

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  1. Weizhen Ye
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  2. Georges Mairet-Coello
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  3. Emanuel DiCicco-Bloom
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Correspondence to Emanuel DiCicco-Bloom.

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Ye, W., Mairet-Coello, G. & DiCicco-Bloom, E. DNAse I pre-treatment markedly enhances detection of nuclear cyclin-dependent kinase inhibitor p57Kip2 and BrdU double immunostaining in embryonic rat brain. Histochem Cell Biol 127, 195–203 (2007). https://doi.org/10.1007/s00418-006-0238-6

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