Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Summary

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Abstract

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae. In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies. The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins. Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Protocol

The following protocol for polytene chromosome squash preparation is adapted from the procedure described in Johansen et al. (2009).

1. Culture of third instar Drosophila larvae

In order to obtain optimal polytene chromosomes for high quality squash preparations, uncrowded culturing conditions are essential (i.e., place around 20 egg-laying female flies in a standard 4" fly bottle and change to a new bottle each day). Select the fattest individuals from the first crop of climbing 3rd instar larvae while they are still wandering but just prior to pupation. We routinely culture at 21°C but 18°C wi....

Discussion

The inclusion of acetic and lactic acids in conventional squash fixation protocols facilitates both interband resolution and chromosomal arm spreading but unfortunately some epitopes do not survive this treatment. An example of such an epitope is H3S10ph (Cai et al., 2008). Since acid treatment also has the disadvantage that it quenches the inherent fluorescence of GFP-tagged proteins, DiMario et al. (2006) recently developed a formaldehyde-based "acid-free squash technique" that allow for direct visual.......