5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

Chippalkatti, Rohan; Suter, Beat (2021). 5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells. Journal of visualized experiments(171) MYJoVE Corporation 10.3791/62642

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In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) has been utilized to investigate replicative/S-phase progression, whereas antibodies against phospho-histone H3 have been utilized to mark mitotic nuclei and cells. A combination of both labels would enable the classification of G0/G1 (Gap phase), S (replicative), and M (mitotic) phases and serve as an important tool to evaluate the effects of mitotic gene knockdowns or null mutants on cell cycle progression. However, the reagents used to mark EdU-labelled cells are incompatible with several secondary antibody-fluorescent tags. This complicates immunostaining, where primary and tagged secondary antibodies are used to mark pH3-positive mitotic cells. This paper describes a step-by-step protocol for the dual-labeling of EdU and pH3 in Drosophila larval neural stem cells, a system utilized extensively to study mitotic factors. Additionally, a protocol is provided for image analysis and quantification to allocate labeled cells in 3 distinct categories, G0/G1, S, S>G2/M (progression from S to G2/M), and M phases.

Item Type:

Journal Article (Further Contribution)

Division/Institute:

08 Faculty of Science > Department of Biology > Institute of Cell Biology

UniBE Contributor:

Chippalkatti, Rohan and Suter, Beat

Subjects:

500 Science > 570 Life sciences; biology

ISSN:

1940-087X

Publisher:

MYJoVE Corporation

Funders:

[42] Schweizerischer Nationalfonds ; [UNSPECIFIED] Universität Bern

Language:

English

Submitter:

Beat Suter

Date Deposited:

26 May 2021 09:52

Last Modified:

26 May 2021 09:52

Publisher DOI:

10.3791/62642

Related URLs:

PubMed ID:

34028440

URI:

https://boris.unibe.ch/id/eprint/156213

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