In FISH the purpose of aging is (1) to fix the biologic material to the glass surface and (2) to increase the "hardness" of the chromosomes, making their structure resistant to the subsequent DNA denaturing.
1. Dry heat
Prior to common cytogenetic banding procedures, slides are usually aged either by incubating 2-3 days at RT, or overnight at 65° C or by heating 30 minutes at 90° C. Same slides can be used for FISH, but results are usually not as good as with fresh slides. However, aging has to be gentle enough to allow optimal access of the labeled DNA probe to the target chromosomal DNA. Very fresh slides, if not aged, will either lose most of the nuclei/chromosomes during denaturing or the shape of the chromosomes will become very distorted (= "puffy" chromosomes). Too long aging will sharply decrease the efficiency of hybridization, which is detrimental especially in CGH or chromosome painting analyses. For example, it was nearly impossible to obtain FISH results on slides older than 2 years, when stored at room temperature. By comparison, 10 year-old cell suspensions stored in fixative or ethanol and kept in a refrigerator or freezer worked well for FISH.
2. Chemical aging (comparison of alcohos)
Chemical aging was developed as an alternative to dry-heat aging, in order to shorten the FISH protocol and increase hybridization efficiency. Its main purpose is to preserve the shape of the chromosomes and allow a good DAPI banding pattern. In chemical aging, chemical reagents (primarily alcohols) not dry heat are used to achieve fixation and shape-preservation of the nuclei/chromosomes on the slide. The simple installation required for chemical aging is depicted here. In a comparison test, slides from the same cell suspension were subjected to aging treatment with methanol, ethanol, isopropanol, butanol, fixative (3:1 methanol: acetic acid) or 1% formaldehyde at room temperature (RT), 65° C or 94° C for various amounts of time (from 1 second to 10 minutes). Alcohols were chosen with the idea that they would replace the water in cells, and allow heating of proteins without changing chromosome morphology. Formaldehyde was selected because it promotes covalent links between aminoacid residua in proteins. Jars containing these solutions, were covered to prevent evaporation, and were kept in different waterbaths at the desired temperatures. To assess the efficiency of every chemical used, two types of tests were performed : (1) hybridizations, using a variety of probes, and (2) DAPI staining. Chemically aged slides were compared among themselves (different chemicals) and with slides subjected to "classical" aging, which uses dry heat.
Examples of some of these tests are shown in Fig. 1g-l. Non-aged slides were pretreated in pepsin (Fig. 1g) or trypsin (Fig. 1i), then denatured. Hybridization worked well after both proteases, but chromosome architecture was not optimal (chromosomes were thick, puffy, unevenly stained by DAPI). Other slides were subjected to 2 minutes chemical aging in ethanol, then pretreated with pepsin (Fig. 1h and 1k) or trypsin (Fig. 1j and 1l), then denatured and hybridized. Chromosome architecture was preserved better after ethanol aging, even after protease treatment. Similarly, nuclei were more evenly stained by DAPI (Fig. 1l) after chemical aging. Nuclear architecture was better preserved by chemical aging than by 2 hour room temperature drying (Fig. 1f).
Other results from these experiments are shown in Fig. 3. Slides were kept 3 minutes at 94 C in the corresponding chemical reagent or in dry heat, then pretreated in trypsin for 10 seconds. Some slides were denatured (Fig. 3g-k) and some were not (Fig. 3a-f), then all slides were stained with DAPI or Giemsa, to compare the preservation of chromosomal architecture. Very few differences were detected on the non-denatured slides. Differences became more obvious after denaturing (2 minutes at 75° C, in 70%FA/2xSSC). When using dry heat, chromosomes changed their appearance, and became "ghost-like" structures after denaturing (Fig. 3k), compared to non-denatured slides (Fig. 3e). Metaphases aged in methanol, ethanol and formaldehyde did not change much after denaturing (Fig. 3g, 3h and 3j). Aging in formaldehyde decreased or prevented hybridization and was abandoned. Ethanol is much less toxic than methanol and evaporates slower, making it much more convenient to use than methanol. Chemical aging worked with 3:1 methanol:acetic acid as well (Fig 2i), but chromosomes became somewhat "wider".
Therefore, methanol, ethanol and regular fixative remained the best candidates for further testing.
3. Why ethanol?
To compare them, slides aged in methanol, ethanol or fixative were subjected to dry heat, protease pretreatment and DAPI staining in various steps (Fig. 4). Our main purpose was to find the best combination allowing both good DAPI banding pattern and good hybridizations. In Fig. 4., thefirst columnshows slides aged in ethanol, thesecond columnshows slides aged in methanol and thethird columnslides aged in fixative. In thefirst row, slides were subjected first to dry heat, then chemical aging and then protease pretreatment. In thesecond row, chemical aging was followed by dry heat and protease treatment. In the third row, dry heat was followed by protease treatment and chemical aging. In thefourth row, chemical aging was followed by protease treatment and dry heat. In all twelve images, the last step was slide denaturing in 70% FA/2x SSC at 75 C, followed by DAPI staining.
Slides in the first and second row show almost no differences, indicating that, if a combination of dry heat and alcohol aging are used before protease pretreatment, slides looked similar. Differences appear between slides aged in ethanol (Fig. 4a, 4d) and slides aged in methanol (Fig. 4b, 4e). Methanol aging resulted in wider, more puffy chromosomes (second column). Dry heat followed by protease pretreatment (third row of images) resulted in more distorted, puffier chromosomes than alcohol aging followed by protease pretreatment (last rows of images). This showed that alcohol aging preserved better chromosome morphology than dry heat. Chromosomes in the last row had somewhat better architecture than chromosomes in any of the other three rows of images. This suggested that chemical aging by itself was sufficient for slide treatment, and did not require additional dry heat to preserve chromosome morphology. As dry heat was found in our experience to be detrimental to the DNA hybridization, it was eliminated completely from the aging process.
Although most of these chemicals worked, because acetic acid is corrosive and methanol is toxic, we chose ethanol for slide aging.
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Last modified on: Feb12, 2001