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.
Bibliographical noteFunding 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.
- Antibody labeling
- Chromatin structure
- Fixation conditions
- Pol II
- Polytene squash preparations