Polytene chromosome squash methods for studying transcription and epigenetic chromatin modification in Drosophila using antibodies

Kristen M. Johansen; Weili Cai; Huai Deng; Xiaomin Bao; Weiguo Zhang; Jack Girton; Jørgen Johansen

doi:10.1016/j.ymeth.2009.02.019

Polytene chromosome squash methods for studying transcription and epigenetic chromatin modification in Drosophila using antibodies

Kristen M. Johansen, Weili Cai, Huai Deng, Xiaomin Bao, Weiguo Zhang, Jack Girton, Jørgen Johansen

Biology (Duluth)

Research output: Contribution to journal › Review article › peer-review

41 Scopus citations

Abstract

The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable antibody labeling and preservation of high resolution chromatin structure.

Original language	English (US)
Pages (from-to)	387-397
Number of pages	11
Journal	Methods
Volume	48
Issue number	4
DOIs	https://doi.org/10.1016/j.ymeth.2009.02.019
State	Published - Aug 2009

Bibliographical note

Funding Information:
We thank members of the laboratory for discussion, advice, and critical reading of the manuscript; Ms. V. Lephart for maintenance of fly stocks; and Mr. Laurence Woodruff for technical assistance. We thank Drs. M. Kuroda, R. Kelley, P. DiMario, and A.S. Belmont for advice and technical suggestions and for providing their protocols prior to publication. This work was supported by National Institutes for Health Grant (GM62916) and National Science Foundation grant (MCB0817107) to K.M.J.

Keywords

Antibody labeling
Chromatin structure
Drosophila
Fixation conditions
Pol II
Polytene squash preparations
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10.1016/j.ymeth.2009.02.019

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title = "Polytene chromosome squash methods for studying transcription and epigenetic chromatin modification in Drosophila using antibodies",

abstract = "The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable antibody labeling and preservation of high resolution chromatin structure.",

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note = "Funding Information: We thank members of the laboratory for discussion, advice, and critical reading of the manuscript; Ms. V. Lephart for maintenance of fly stocks; and Mr. Laurence Woodruff for technical assistance. We thank Drs. M. Kuroda, R. Kelley, P. DiMario, and A.S. Belmont for advice and technical suggestions and for providing their protocols prior to publication. This work was supported by National Institutes for Health Grant (GM62916) and National Science Foundation grant (MCB0817107) to K.M.J.",

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AU - Johansen, Kristen M.

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AU - Deng, Huai

AU - Bao, Xiaomin

AU - Zhang, Weiguo

AU - Girton, Jack

AU - Johansen, Jørgen

N1 - Funding Information: We thank members of the laboratory for discussion, advice, and critical reading of the manuscript; Ms. V. Lephart for maintenance of fly stocks; and Mr. Laurence Woodruff for technical assistance. We thank Drs. M. Kuroda, R. Kelley, P. DiMario, and A.S. Belmont for advice and technical suggestions and for providing their protocols prior to publication. This work was supported by National Institutes for Health Grant (GM62916) and National Science Foundation grant (MCB0817107) to K.M.J.

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N2 - The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable antibody labeling and preservation of high resolution chromatin structure.

AB - The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable antibody labeling and preservation of high resolution chromatin structure.

KW - Antibody labeling

KW - Chromatin structure

KW - Drosophila

KW - Fixation conditions

KW - Pol II

KW - Polytene squash preparations

KW - Transcription

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Polytene chromosome squash methods for studying transcription and epigenetic chromatin modification in Drosophila using antibodies

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