Advancing Epigenetics Towards Systems Biology

Preparation and Immunostaining of Polytene Chromosome Squashes (Prot 1)

Christine James, Paul Badenhorst & Bryan Turner

Introduction

The polytene chromosomes found in the salivary glands of Drosophila larvae (and other diptera), provide a valuable model system in which microscopical techniques can be used to study the functioning of the interphase genome. Chromosome banding patterns, revealed by simple DNA stains or phase contrast, provide markers by which individual chromosomes can be identified and by which specific genes or genomic regions can be located. Immunostaining provides an additional level of resolution by allowing non-histone proteins or modified histones to be located to such genomic regions, or associated with specific chromatin functions (eg. transcription) or chromatin types (e.g. heterochromatin).

However, procedures that give the best polytene chromosome preparations (squashes) with the most clearly-resolved bands, involve treatment with concentrated acetic acid. If unmodified, such procedures result in extraction of all, or nearly all, the histones and most non-histone proteins. To prevent this, it is necessary to pre-fix the chromosomes with formaldehyde. Unfortunately, such fixation compromises the spreading of polytene chromosomes. A squashing procedure that allows successful immunolabelling requires a fine balance between fixation that is sufficient to retain a high enough proportion of the protein of interest, while not preventing the preparation of suitably spread and banded chromosomes. We have found the following procedure to be successful for squashing and immunolabelling with several different antisera to modified histones H3 and H4.

PDF version

Christine James, Paul Badenhorst & Bryan Turner

Institute of Biomedical Research
University of Birmingham Medical School
Birmingham
B15 2TT, UK

Christine James, Paul Badenhorst & Bryan Turner