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Topics: 1.Preparation 2.Aging 3.Denaturing 4.Methods 5.Table | Fig1 | Fig2 |
FISH: chemical aging.
Aging of cytogenetic preparations denatures the proteins, removes water and fixative and enhances the adherence of the material to the glass. When fresh, non-aged slides are heat-denatured, they either lose most of the material or chromosomes become very distorted ("puffy"). If slides are extensively aged, hybridization efficiency decreases because the chromosomes are too "hard". Chemical aging was developed as an alternative to dry-heat aging, in order to shorten the FISH protocol and preserve cell freshness (thus increasing hybridization efficiency) and chromosome architecture (thus allowing DAPI staining). Chemical reagents (usually alcohols) were used to achieve fixation. In a "chemical test", identical slides were subjected to heat-pretreatment with methanol, ethanol, isopropanol, butanol, fixative (3:1 methanol: acetic acid) or 1% formaldehyde at 37Ú, 65Ú C or 94Ú C for various amounts of time (from 1 second to 10 minutes, data not shown). Hybridization results and DAPI staining on all slides indicated that heat-treatment in fixative or ethanol at 94Ú C for 2-20 seconds worked best. Ethanol was preferred, because of its lesser toxicity. The better quality of slides treated by chemical aging translates itself into shorter hybridization times: 15-20 minutes for centromeres (Fig. 2g), 4-5 hours for single copy probes such as cosmids (Fig. 2i), and overnight hybridization for complex probes, as for CGH (Fig. 2j-k) or M-FISH (Fig. 2l).
FISH: pepsin pretreatment.
Regardless of the aging procedure, slides subjected to protease pretreatment prior to denaturing, always showed improved hybridization results (Fig. 2e-f). 0.005% pepsin in 0.01N HCl is convenient because its action is pH-dependent and can be immediately stopped by rinsing the slide in a buffer at pH 7-8. Incubation time in pepsin depends on the aging process. Slides chemically aged for 2 minutes require longer pepsin treatment (1-2 minutes) than slides chemically aged 10 seconds (30 seconds pepsin incubation) to yield similar hybridization results and DAPI banding. By comparison, slides dry-heated overnight at 65Ú C (as for G-banding) require 10-15 minutes pepsin treatment, and, although DAPI bands are sharper hybridization quality decreases.