1. The hybridization procedure is very similar to protocols to used with metaphase spreads. In the hybridization mix, combine 1 ^L of each probe, 1 ^L of human COT1™ DNA (1 ^g/^L, Invitrogen; optional), 1 ^L of salmon sperm DNA (5 Prime-3 Prime, Boulder, CO), and 7 ^L of MM2.1.

2. Fiber hybridizations include a comparatively low concentration of a biotin- or FITC-labeled DNA probe prepared from the high molecular weight DNA that is used to prepare the fibers. This counterstain highlights the otherwise invisible DNA fibers and allows competitive displacement by the probes to be mapped along the DNA fiber (51,56). Additionally, one or several cloning vector-specific probes are included to allow determination of the orientation of the insert (60).

3. Apply the hybridization mixture to the slides and cover-slip. Avoid bubbles; if bubbles occur, try to squeeze them out gently with fine-tip forceps, avoiding movement of the cover slip.

4. Transfer the slides to a dry bath (or "hot plate") and denature the DNA at 88°C for 90 s.

5. Transfer the slides to a moisture chamber (a plastic or stainless steel box with a wet paper towel at the bottom and support such as cut disposable plastic pipetors to raise the slide) and incubate it overnight at 37°C.

6. The wash and detection steps are not much different from protocols used for FISH to interphase and metaphase cells and have been described in detail (51,54). After hybridization, the slides are washed three times in 2X SSC at 20°C for 10 min each and then incubated with 100 ^L PNM buffer or blocking stock solution under a plastic cover slip at 20°C for 5 min (see Note 12). The slides are then incubated at room temperature for 30 min with 100 ^L PNM buffer containing AMCA-avidin (Pharmacia), anti-digoxigenin-rhodamine (Roche), and a mouse antibody against FITC (DAKO) (see Note 13).

7. The slide is then washed two to three times in 2X SSC for 15 min each at 20°C with constant motion on a shaking platform.

8. If necessary, signals are amplified using a biotinylated antibody against avidin raised in goat (Vector) followed by another layer of AMCA-avidin, a Texas Redlabeled antibody against sheep raised in rabbit, and a horse-anti-mouse antibody conjugated to FITC (Vector) (52).

9. The slide is mounted in 8 ^L of antifade solution and covered by a 22 o 22-mm cover slip.

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