Fluorescent in situ Hybridization Combined with Immunostaining on Polytene Chromosomes (Prot 4)
Polytene chromosomes are present in many larval tissues in Drosophila. They result from subsequent rounds of DNA replication not followed by cell division, and they represent a material of choice to determine by cytological methods whether a particular DNA sequence is associated with a protein of interest. They can be most easily prepared from salivary glands of third instar larvae, where the degree of polytenization is maximal.
We previously described a method that combined protein immunostaining with fluorescent in situ hybridization (FISH) in order to allow simultaneous visualization of a protein of interest and a specific DNA sequence (Dejardin and Cavalli, 2004; Lavrov et al., 2004). Compared with previous methods, where FISH and immunostaining were performed in separate experiments, this approach has the great advantage of directly showing the co-localization of a protein with the locus of interest. However, this combination raises a number of technical difficulties. In some cases, the antibody immunostaining does not survive the FISH procedure. In the published protocol, we provided a set of solutions for this problem. In the worst situation, where the immunostaining signal can not preserved from degradation during FISH, one has to perform immunostaining, acquire many chromosome images under conditions in which it is possible to keep the record of the XY position of the microscope stage, then perform FISH, find the same chromosomes by repositioning the slide in the same XY coordinates as for immunostaining, acquire the FISH images, and finally superimpose the immunostaining and the FISH images using software such as Adobe® Photoshop® (Lavrov et al., 2004). Although this procedure is straightforward, it is quite labor intensive. [...]
Institute of Human Genetics
CNRS UPR 1142 - 141, rue de la Cardonille - 34396 Montpellier cedex 5, France
Immunofluorescence and Fluorescent In Situ Hybridization in Mouse Fibroblasts and Embryonic Stem Cells (Prot 8)
Techniques routinely used in our lab for analysing the X-inactivation process in differentiating ES cells are described. We focus particularly on chromatin changes (histone modifications, protein association...) during X inactivation, using immunofluorescence (IF) combined with RNA FISH or DNA FISH on interphase nuclei. These should provide a tool for defining potential causal relationships between different events not only during X inactivation but also during the establishment of other patterns of gene activity.
The main purpose of a combined IF and FISH analysis is, on the one hand, to preserve nuclear architecture and the antibody's epitope as far as possible but, on the other hand, to allow the penetration of the FISH probe for detection of nuclear transcripts, gene location or chromosome territories. The optimal conditions for IF are usually poorly compatible with those for FISH. We have therefore tested a variety of methods and conditions and we describe here those that we find optimal for the immuno-detection of histone modifications combined with RNA or DNA FISH on mouse fibroblasts or embryonic stem cells. For more details, the reader is referred to Chaumeil et al., 2002 and Chaumeil et al., 2004.
Mammalian Development Epigenetics Group
UMR 218 Institute Curie - 26, rue d'Ulm - 75231 Paris Cedec 05, France
Competing Chromosomal Proteins from Drosophila Polytene Chromosomes Using Modified Histone Peptides (Prot 2)
This is an adaptation of the published procedure for RNAse treatment of Drosophila polytene chromosomes to remove proteins whose binding is RNA dependent, such as the dosage compensation complex member MLE (Richter et al., 1996). In this adaptation, I used differently methylated histone tail peptides to compete Polycomb protein from polytene chromosomes, finding that competition was peptide specific and locus-specific (Ringrose et al., 2004). (I also tried RNAse treatment, but found it to have no effect on Polycomb binding).
Strictly speaking, this is not an in vivo assay, as the salivary gands are removed from larvae just before peptide competition. However, since the assay is performed on unfixed, intact salivary glands, the hope is that it will reflect the in vivo behaviour of chromatin binding proteins to some extent. Another limitation is that the salivary glands represent a rather strange tissue, that will not persist in the adult fly, and whose chromosomes have undergone many rounds of endoreplication. However salivary gland chromosomes are unparalleled for cytological purposes, and thus have many advantages if specific sites of competition of chromatin binding proteins are to be mapped (see Ringrose et al., 2004). [...]
Institute of Molecular Biotechnology (IMBA) GmbH
Dr. Bohr-Gasse 3 - 1030 Vienna, Austria
Preparation and Immunostaining of Polytene Chromosome Squashes (Prot 1)
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.
Institute of Biomedical Research
University of Birmingham Medical School
B15 2TT, UK
Preparation of extended chromatin fibers from human tissue culture cells (Prot 55)
Extended chromatin fibers can be used to examine the linear structure of chromatin by high-resolution immunofluorescent microscopy. Previous applications include studies of the distribution of canonical histone H3 chromatin and centromeric histone CenH3 chromatin at centromeres (Blower et al, 2002; Dunleavy et al, 2011; Sullivan & Karpen, 2004). This technique can be combined with fluorescent in situ hybridization (FISH) for specific DNA sequences e.g. alpha satellite arrays at centromeres (Blower et al, 2002; Lam et al, 2006) or with the incorporation of thymidine analogs (BrdU, EdU) to mark newly replicated DNA (Blower et al, 2002; Dunleavy et al, 2011).
Postdoctoral Fellow (Karpen Laboratory), Lawrence Berkeley National Laboratory, Department of Genome Dynamics, 1 Cyclotron Road, MS977, Berkeley, CA 94720, USA
Corresponding author: Elaine Dunleavy