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FISH = Materials/Methods
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Principle
In fluorescence in situ hybridization (FISH), a DNA probe is labeled with a fluorescent dye or a haptene (usually in the form of fluor-dUTP or haptene-dUTP, that is incorporated into the DNA using enzymatic reactions, such as nick translation or PCR). The labeled DNA is purified, concentrated, resuspended in hybridization buffer (containing formamide) and is hybridized onto chromosomes and nuclei on slides (cytogenetic preparations). After overnight hybridization, the slides are rinsed in washing solutions and, if needed, one or several layers of fluorescent-labeled antibodies are added to detect haptene-labeled DNA. The slides is mounted with antifade solution and is visualized at the fluorescent microscope, using appropriate filters. This procedure is illustrated in the figure below.
Cell type considerations (for FISH)
Due to the ease in culturing and to the high metaphase index, lymphocytes from peripheral blood cultures are preferred for use in most laboratories. 30-40 minutes colcemid treatment of 48-72 hour cultures can yield a high metaphase index, with remarkably similar chromosome lengths, very useful particularly for CGH studies. Fibroblasts can also be easily used for general FISH purposes (probe mapping, interphase FISH). The metaphase index is, in general, lower than for fresh lymphocyte preparations. There is a larger variability in chromosome sizes among metaphases of the same pellet, due primarily to longer colcemid times. 4-8 hours colcemid treatment on fibroblast cultures can significantly increase the metaphase index, but many of the metaphases will have very short chromosomes. In general, the chromosomes of fibroblast cultures overspread very easily on the slide, indicating an increased fragility of the cell membranes with usual fixative treatment. Lymphoblastoid and tumor cell lines are more difficult to use in FISH analyses, as the metaphase index is lower and the shape/spread of the chromosomes is more difficult to control.
Slide preparation.
(detailed here)
Chemical aging, pretreatment and hybridization.
(detailed here)
Long-term storage
For good FISH results on older cytogenetic slides, the slides should not be stored dry at room temperature. Instead, slides should be stored either in 100% ethanol at -20° C, or should be placed in a plastic box wrapped up in Saran Wrap, and stored at -20° or -80° C. With any of these procedures, slides can be stored for years and can be used in FISH without major problems.
Visualization of results.
Over the duration of this work (4-5 years), there were several ways in which pictures were taken. Some of the images were captured directly on photographic film for transparencies (Kodak Ektachrome 400) using a photographic camera (Leica) attached to a fluorescent microscope (Leica Aristoplan). Other images were taken using a cooled CCD camera (Photometrics) attached to a Leica Aristoplan microscope and a commercial software package (Vysis, Inc) running on a Macintosh 8500 computer. Yet other images (mostly M-FISH) were captured using a cooled CCD camera (Sensys or Quantix-Photometrics) attached to an Olympus microscope and another commercial software package (Perceptive Scientific Instruments PSI) running on a Macintosh G3 system.
Probes, labeling procedures, analysis of results.
Table
1. Nick translation and PCR labeling.
Labeling was done using either fluorescent (direct)-or haptene (indirect)-labeled
nucleotides.
Commercially-labeled nucleotides used throughout this work are shown
in Table
2.
See a more thorough discussion on how to prepare your own, custom-labeled
nucleotides here.
Briefly, reactive aminoallyl-dUTP and succinimidyl-ester derivatives
of many dyes can be chemically coupled in a simple reaction, in the
presence of bicarbonate (alkalyne pH). After the reaction, the nucleotides
can be used directly for enzymatic DNA labeling reactions, if BSA (bovine
serum albumin) is added to the mixture (probably to block free radicals).
After labeling, the DNA MUST be purified, in order to remove especially
the residual free dye. Using custom-made fluor-dUTPs decreases significantly
the costs of the FISH analysis. A schematic description of the process
is depicted in the figure below.
Antibody detection was performed using a wide variety of labeled antibodies (Table 3). Antibody use is detailed here.
All antibodies used were IgG molecules raised against other whole IgG molecules. Detection schemes were chosen carefully, to prevent unwanted antibody-antibody interactions. Antibodies were purchased, labeled or non-labeled, from the following vendors: Sigma; Accurate Chemical and Scientific Co.; Vector Laboratories; Boehringer Mannheim (Indianapolis, IN); Molecular Probes. Antibodies were also labeled in the laboratory using common protein labeling protocols (Molecular Probes). Reactive dyes for antibody labeling were purchased from Pharmacia-Amersham or Molecular Probes.