Chromosome mounting

Alternatively, DNA on the slide can be hybridized with probe, followed by staining to effect chromosome banding. Fluorescence of the hybridized region can be photographed and mounted to serve as a permanent record. 

Add 60 xL of DAB substrate solution and leave for 5 min at room temperature. Rinse the slides in tap water to stop the reaction (j Note 10). Counterstain for 5 min in 10% Giemsa, rinse in buffer, and air-dry (se Note 11). Permanent mounting without subsequent foding of the chromosome and/or hybridization signal is best achieved by mounting slides in Xam. 

To generate probes that are suitable for hybridization to chromosomes in whole-mount specimens, several factors must be considered. Both the size of the target genomic sequence and the fragment length of probe molecules are crucial for success. 

For hybridization to whole-mount tissues, probe fragment size should be carefully monitored and kept below an average length of 150 bp. This size restriction is important to allow probes to penetrate and diffuse freely within the tissue. For chromosomes fixed with acid/alcohol and spread on slides, this is not critical probe fragments up to a few kilobases in size are acceptable, however, because most of our probes are eventually used for whole-mount experiments, we use this procedure for all probe synthesis. 

Du Praw, E. J. 1965. Macromolecular organization of nuclei and chromosomes A folded fibre model based on whole-mount electron microscopy. Nature (London), 206 338-343. 

H. Gardner and H. Punett, An improved squash technique for human male meiotic chromosomes Softening and concentration of cells mounting in Hoyer s medium. Stain Technol. 39, 245-248 (1964). 

Dernburg A.F. 1999. Fluorescence in situ hybridization in whole-mount tissues. In Chromosome structural analysis A practical approach (ed. W.A. Bickmore). Oxford University Press, United Kingdom. (In press.)... 

Much has been learned about Drosophila meiosis by immunocytochemical studies of whole-mount oocytes lacking functional meiotic proteins and by localization of some of the relevant proteins (McKim et al. 1993 White-Cooper et al. 1993 Afshar et al. 1995 Endow and Komma 1996 Matthies et al. 1996 Moore et al. 1998). However, none of these classic methods easily point out kinetic problems, nor are the earliest points of deviation always readily obvious by static methods. For these reasons, and because the behavior of the highly dynamic components of the meiotic spindle may be perturbed in meiotic mutants, methods have been developed to directly observe the cytoskeleton and chromosomes in living egg chambers (Theurkauf 1994a Endow and Komma 1996 Matthies et al. 1996). 

Note Giemsa stain wiU fade within a few hours. However, chromosomes can be washed in PBS and restained. For storage, slides can be frozen at -20 C. Entelan (EM Science) can be used as a permanent mounting solution. 

The general strategy for FISH is to equilibrate the tissue in buffered formamide, to add the probe(s), and to denature both the chromosomal DNA and probe together by heat treatment. The probe is then allowed to anneal for several hours at an appropriate temperature, unbound probe is washed away, and secondary detection (if required) is performed. The nuclear DNA is then usually counterstained, and the sample is mounted for microscopy. For experiments in which immunolocalization of other cellular components is desired, these staining steps can conveniently be performed after hybridization. 

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