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Cell suspension quality
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We studied how the nuclear or chromosomal morphology of two non-related cell suspensions responds to the various steps of the FISH protocol (Fig. 2g-l). Simple Giemsa staining was used to quickly test the quality of the slides, as it was faster and easier than DAPI staining or hybridization. One cell suspension, probably sub-optimally treated with hypotonic buffer, showed more cytoplasmic residua around metaphases and nuclei. Slides from this suspension, were subjected to various times of ethanol aging, 10 seconds in Fig. 2g, 50 seconds in Fig. 2h and 2 minutes in Fig. 2i, and were denatured, treated with trypsin and G-banded. The G-banded pattern, although not very sharp, improved somewhat along with increasing times of ethanol aging. In general, the use of protease after denaturing was not very useful.
A second cell suspension, showing much less cytoplasmic residua was used to prepare another set of slides. One of these (Fig. 2j) was subjected to 2 minutes ethanol aging followed by trypsin and Giemsa staining, but without denaturing. The second slide (Fig. 2k) was ethanol aged 2 minutes, denatured and Giemsa stained. The third slide (Fig. 2l) was subjected to 60 minutes dry heat (94 C) followed by Giemsa staining. A chromosome banding pattern could be distinguished on all slides, but banding was best when dry heat was used (Fig. 2l), and worse after denaturing (2j better than 2k).
In Fig. 2i and 2k, slides prepared from the two suspensions were treated identically and were compared. Chromosomes in Fig. 2k, which belonged to the cells with less cytoplasmic residua, showed better morphology and banding pattern. Thus, proper preparation of cell suspensions and optimal hypotonic buffer pretreatment are important for both banding and FISH procedures.
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