For comparison purposes, the same cell suspension was used in all experiments describing the slide preparation procedure. The cells were resuspended in 1:1, 3:1, 6:1 methanol:acetic acid or 3:1 ethanol acetic acid fixative(s) and slides were prepared using various changes (described for each figure) of a basic protocol. In the basic protocol, cell suspension was placed on dry, commercially pre-cleaned slides, which were then subjected or not to water vapors and dried on the metal plate of the waterbath at various temperatures.
Tissue culture and harvesting
Peripheral blood culture was performed according to common protocols, by inoculating sodium heparin collected whole blood in 1640 RPMI/10% FCS and PHA stimulation. 72 hours cultures were subjected to colchicine (Colcemid, Life Technologies, Gaithersburg, MD, USA) for 40 minutes, and the cells were then resuspended in 0.075M KCL hypotonic buffer for 15 minutes at 37? C. A small volume of 3:1 methanol:acetic acid fixative was added to the cell suspension and 3 more fixative washes were subsequently performed. Cell pellet(s) were stored at -20? C in fixative.
The best cell suspension was chosen for comparative studies. These cells had a high metaphase index and were of very good quality (few debris).
Equipment (fig. 1)
This includes a waterbath in which the water has been brought to 75-80? C. A waterbath with recirculating capabilities close to the water surface is more desirable, as the amount of steam produced is higher, but any waterbath will work. It is important to bring the water level to within 1-2 cm or so from the top. This will allow heating of a flat metal surface (the lid or any metal plate) close to the temperature of the water. The metal plate is placed on top of the waterbath so as to cover it only partially, leaving an opening of about 5-10 cm wide through which hot steam comes out. At the other end of the metal plate, an area about 10 cm wide (at least) should "hang" in the air and not be in contact with the steam. If a 2-3mm thick metal plate is used, this setting allows a smooth temperature gradient across the metal plate surface, from 70? C or so at the "hot" end, facing the water, to about the room temperature at the "cold" end of the plate. Too thin metal plates bend too easily, whereas too thick plates have a lower temperature gradient.
Slide preparation was done in a common laboratory, equipped with the setting described (fig.1). Briefly, 20-25 uL cell suspension were placed on a slide taken directly from its original package, using a common automatic pipette. Dropping from a height was not necessary. The suspension was spread evenly by moving gently the pipette tip parallel to the surface. Excess liquid was drained on a paper towel. When the surface of the slide became grainy (as the fixative evaporated), the slide was placed face-down in the steam of a hot waterbath for 1-3 seconds, then was dried by placing it on a metal plate (carrying a gradient of temperature across its surface). The hot steam overpowered any potential influence of the atmospheric humidity. Drying temperature depended on the pellet quality and NOT on the climate, and could be adjusted at will, to achieve any degree of spreading.
Slides were examined under phase using a Leica Aristoplan or an Olympus microscope, with a 10x or 20x objective. Serial pictures during the drying process and of the unstained slide preparations (fig. 3, 4) were taken using an Olympus microscope equipped with a CCD camera (Perceptive Scientific Instruments).
G-banding was performed according to standard laboratory procedures. Prior to banding, slides were incubated overnight at 65? C. For staining, slides were incubated 3 seconds to 1 minute in a trypsin (DIFCO 1:250) solution (0.1g Trypsin in 100 ml isotonic buffer). Slides were rinsed for a few seconds in a jar with FCS (2-3 ml FCS in 50 ml isotonic buffer) and then rinsed in isotonic buffer and incubated for 1.5-5 minutes in a Coplin jar with Giemsa stain (EM Science). Giemsa solution was prepared by mixing 1.5 ml Giemsa with 50 ml Gurr’s phosphate buffer [1 tablet Gurr’s (BDH Lab Supplies) in 1000 ml water]. After staining, slides were rinsed a few seconds each in Gurr’s buffer and water and then air-dried. Slides were examined with a Leica Aristoplan and images were taken using a cooled CCD camera (Sensys, Photometrics) and specially designed software (Vysis, Inc). Images shown (fig. 5, 6) do not reflect the best banding protocol or staining pattern that can be obtained. Instead, these images are intended to illustrate the way in which changing various conditions in the protocol or slide preparation influences the quality of banding.
Fluorescence in situ hybridization (FISH)
Chemical aging. (Note: ethanol is used to achieve fixation and shape-preservation of the nuclei/chromosomes on the slide). Place slide on the metal block of a thermocycler (a PCR machine, Fig.1b), pipette 150-200 µl ethanol on the slide, cover with a coverslip and cover the slide/coverslip assembly with ethanol-soaked gauze, to prevent ethanol evaporation. Program the block to increase its temperature to 94? C, hold for 10-20 seconds, then cool towards room temperature. Depending on the machine, the heating and cooling speed is 1-2? C/second. Alternatively, incubate the slide 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. Gradual thermal treatment works better than sudden temperature changes for chromosome shape preservation. To harden the chromosomes more and ensure sharper subsequent DAPI banding, the time the slides are incubated at 94? C in ethanol can be increased up to 2 minutes.
Pepsin pretreatment.In a jar, add 49.5 ml water, 0.5ml 1N HCl and 25-30ul 10% (w/v) pepsin and incubate 10-15minutes at 37? C prior to use (epsin solution has a concentration of 0.005%). Slides chemically aged for 10-15 seconds at 94? C will be incubated in pepsin solution 30-60 seconds, rinsed briefly in PBS and incubated 5 minutes each in 70% and 100% ethanol, followed by air-drying. Slides aged longer (2 minutes at 94? C) should be subjected to 1-2 minute pepsin pretreatment.Overnight baked slides were pretreated in pepsin for 10-12 minutes.Invariably, pepsin pretreated slides produced better FISH signals compared with non-pretreated slides, regardless of how they were prepared or aged.
Slide denaturing(2 alternative procedures):
Separate denaturing (Fig. 2i, 2l).Place 150ul of 70%formamide/2xSSC on slide, cover with a 50x22mm coverslip and place slide on the metal block of the thermocycler. Denature as described above. When reaching safe temperatures, pick up the slide, let coverslip slide off and place slide 3 minutes each in 70% and 100% ethanol at room temperature, then air-dry. The probe is denatured by keeping the vial 5 minutes at 75? C followed by cooling at room temperature. Probe pre-annealing is not essential but helps reduce background somewhat. Place 11-12ul probe on the slide, cover with coverslip, seal with rubber cement and incubate for the desired amount of time at 37? C in moist chamber.