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Topics: 1.Preparation 2.Aging 3.Denaturing 4.Methods 5.Table | Fig1 | Fig2 |
1. EQUIPMENT (Fig. 1a)
The equipment required for the metaphase spreading protocol includes: (1) a source of hot steam (waterbath at 75-80? C); (2) a heated metal plate, ideally with a temperature gradient across its surface. To achieve this, the metal plate/lid covers the waterbath only partially (Fig. 1a), and carries a temperature gradient between the hot end and a cold end. A 2-3 mm thick metal plate is ideal. We tested plates made of stainless steel (3 mm and 1.5 mm thick) and copper (1 mm thick). When the plate rests about 2 cm from the hot water surface, the part of the plate in contact with the hot steam shows roughly 70? C, whereas the other end of the plate (10-15 cm long) is at room temperature. If such a metal plate is not available, a minimum of two surfaces, one at 65? -75? C (a simple heat block) and one at room temperature are required; (3) good quality slides. Two brands of pre-cleaned slides, Gold Seal (Becton, Dickinson Co, Portsmouth, NH) and Superfrost (Erie Scientific, Portsmouth, NH) were commonly used. They did not require any extra cleaning steps or washes prior to use. Cell suspensions were dropped on slides taken directly out of their original boxes. Lower quality glass-slides can be passed through successive washes in acetone, HCl/ethanol and triple distilled water and their quality compared.
Legend: Fig. 1.a: Waterbath image showing metal plate position, allowing both the temperature gradient formation and access to the hot steam; Fig.1b: Chemical aging setting: a few layers of gauze are placed in a plastic box (for example, a box used to store slides ). Two-three stripes of tape are placed across the opening to hold the gauze in place when the box is turned upside-down. Prior to use, the gauze is soaked in ethanol. Two-three freshly prepared slides are placed on the metal block of a thermocycler. 200 ul ethanol are pipetted on each slide, and covered with a coverslip. The plastic box is placed upside-down on the slides, so that the wet gauze comes in contact with the slides and covers them (the gauze will prevent ethanol evaporation during the subsequent heating step). The slides are heated according to the sequence described in the text. After heating, the plastic box/cover and the coverslips are removed and the slides are air-dried.
2. CHROMOSOME PREPARATION.
Cell culture and fixation.
Cell suspensions from peripheral blood, fibroblasts, bone marrows, lymphoblastoid cell lines and germ cell tumors were used to prepare metaphases for G-banding and FISH. Cell culture was performed according to standard protocols. For harvesting, the hypotonic buffer used was 0.075M KCl, at 37° C for 10-20 minutes (incubations were shorter for peripheral blood cultures, longer for tumors). Hypotonic treatment increases cell volume, and disrupts the cell membrane of the red blood cells (allowing their removal).
After hypotonic treatment, usually in 15 ml centrifuge tubes, cells were pelleted by centrifugation 10min at 1000 rpm and are resuspended in 1-1.5 ml fixative (3:1 methanol : acetic acid), then transferred into 1.5 ml vials. All subsequent fixative washes were done in these small vials, with centrifugations performed in a tabletop microfuge for 1-2 minutes at 6-7,000 rpm.
Chromosome spreading and G-banding.
Cells in fixative were diluted at the appropriate density, empirically determined in any cytogenetic laboratory, to produce a reasonable number of well-spread metaphases in each microscopic field. With an automatic pipette, 25-35µl of cell suspension were evenly distributed on several locations on the slide and the liquid was spread by gently moving the pipette tip parallel to the surface. As the fixative gradually evaporated, the surface of the slide became ‘grainy’ (cells visible). At that moment, the slide was placed face down into the steam of the hot water bath (75? C or more) for 1-3 seconds, then dried by placing the slide on the metal plate (carrying a gradient of temperature across its surface, Fig. 1a). The degree of spreading is adjusted using the different temperatures of the heated plate, with higher temperatures increasing chromosome spreading. For difficult to spread cells, after thesurface became ‘grainy’, the slide was passed briefly through the water vapors, then 4-6 droplets of acetic acid were placed on the slide. After the acetic acid slowly spreaded and covered the surface, the slide was held 3-5 seconds in the steam of the waterbath, then quickly dried on the hottest area of the metal plate or the metal block (65? C). After overnight incubation at 65? C (aging), G-banding was performed according to a standard laboratory procedure.
3. FISH: CHEMICAL AGING and SLIDE DENATURING.
Chemical aging.
A freshly prepared slide was placed on the metal block of a thermocycler (a PCR machine, Fig. 1b). 150-200µl ethanol were pipetted on the slide, covered with a coverslip and ethanol-soaked gauze placed on top of them to prevent ethanol evaporation. The block was programmed to increase its temperature to 94? C, keep it for 2-20 seconds, then cool towards room temperature. Depending on the machine, the heating and cooling speed was 1-2? C/second. Alternatively, the slide can be incubated 10-15 seconds each, in jars with ethanol at 50? C, 75? C, 94? C, 75? C and 50? C, followed by drying at room temperature.
After aging, the slides were subjected to brief (30-60 seconds) pepsin pretreatment, using 0.005% pepsin in 0.01N HCl., followed by rinsing in PBS and ethanol series, then air-drying.
Gradual denaturing.
"Simultaneous" protocol:(Fig. 2j-k). 11-12ul labeled DNA probe in hybridization buffer were pipetted on the slide, covered with a 22x22mm coverslip and sealed with rubber cement. The slide was placed on the metal block of a thermocycler, which was programmed to gradually (within 90 seconds) heat the slide to 75? C, keep that temperature for 90-120 seconds, and gradually (90 seconds) cool to room temperature. "Separate" protocol(Fig. 2i, 2l).. Labeled DNA probe is denatured 5 minutes at 75? C in a waterbath. For slide denaturing, 150 µl of 70%formamide/2xSSC are pipetted on the slide, covered with a coverslip and the assembly is gradually heated on the metal block of the thermocycler as described above. Coverslip is discarded and the slide is rinsed through ethanol series, 3 minutes each in 70% and 100% ethanol at room temperature, then air-dried. For laboratories not equipped with a thermocycler, an alternative separate denaturing protocol uses three jars with 70%FA/2xSSC solution at 45? C, 60? C and 75? C. The slide is dipped 5-10 seconds each in the 45? C and 60? C solutions, then incubated 90-120 seconds at 75? C and dipped again 5-10 seconds each in the 60? C and 45? C solutions, followed by brief rinsing in 70%, 100% ethanol and air drying.
Probe labeling, and detection.
Probes (centromeric repeats, chromosome paint probes, cosmids) were either purchased or were prepared and labeled by nick translation or PCR labeling, using commercially available (Boehringer Mannheim, Indianapolis, IN) or custom-made FITC-, biotin- or digoxigenin-dUTP. After overnight hybridization in a moist chamber at 37° C, the slides were subjected to one layer of detection for biotin (avidin-Cy3) and digoxigenin (anti-digoxigenin-FITC). Slides were examined with a Leica Aristoplan or an Olympus Provis AX70 microscope and images taken using cooled-CCD cameras (Photometrics, Tucson, AZ) and commercial software (PSI, Inc or Vysis, Inc).