Hoechst chromosome staining

The following procedure (Gatti et al. 1976 Gatti and Pimpinelli 1983) is a modification of a protocol developed by Latt (1973) for mammalian chromosomes. For other Hoechst-staining protocols, see Holmquist (1975) and Hazelrigg et al. (1982). 

Notes The Hoechst stain is for quality control of the DNA denaturation step. The euchromatin of interphase chromosomes produces a fuzzy Hoechst stain if properly denatured. Heterochromatin and mitotic chromosomes stain fairly well, despite the denaturation. 

Fig. 8.21. A bivariate flow karyotype for human chromosomes stained with Hoechst 33258 and chromomycin A3. From Gray and Cram (1990).

Metaphase chromosomes can be sorted by flow cytometry in the same way that cells can be sorted (see 10.7.5). Usually separation is based on the DNA content of ethidium bromide or propidium iodide stained chromosomes but Hoechst 33258 (which preferentially stains AT rich segments) and chromomycin A3 (which stains CG rich regions) can also be used (Davies et al., 1981). 

Holmquist G. 1975. Hoechst 33258 fluorescent staining of Drosophila chromosomes. Chromosoma 49 333-356. 

Several techniques have been developed for preparation of male meiotic chromosomes (see, e.g.. Cooper 1965 Ashbumer 1989 Miyazaki and Orr-Weaver 1992). All of these techniques allow a clear visualization of chromosomes, but not of other cellular structures such as the cytoskeleton and the spindle. We describe here three of these techniques that in our hands give good reproducible results. The first technique, developed by Lifschytz and Hareven (1977) and described below in Protocol 5.9, is particularly usefid for the analysis of adult testes. The other two techniques (Ripoll et al. 1985 Cenci et al. 1997) are routinely used in our laboratory for larval and pupal testis preparations. The technique described in Protocol 5.10 is a very simple method for preparing aceto-orcein-stained chromosomes. The other technique outlined in Protocol 5.11 allows preparation of unstained chromosomes that can be subsequently stained with various dyes such as Giemsa, Hoechst 33258, and DAPl, or processed for in situ hybridization. 

Chromosomes with fluorescent signals can be counterstained with Hoechst 33258 as described below. However, banding patterns produced by the fluorescent DNA stains are often difficult to compare with the published polytene chromosome-banding patterns. We find it more convenient to simply use phase contrast to correlate the fluorescent signal with the corresponding chromosome bands. 

Staining mitotic chromosomes with Hoechst 33258 Protocol 5... 

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