Fluorescence Microscopy and Visualization

1. To each slide, add 100-300 pL of streptavidin-fluorophore conjugate diluted in TNB buffer; use dilution recommended by manufacturer: streptavidin-Texas red (NEN) is used at 1:500.

2. Place slides in a humidified chamber for 30 min at room temperature.

3. Wash slides in TNT buffer, with agitation, three times for 5 min at room temperature.

4. For staining of chromosomes (counterstain) add two drops of DAPI II and mount for microscopy. Blot excess DAPI, cover, seal the ends of the cover slip with rubber cement, and refrigerate at 4°C for 30-60 min (see Note 12).

4. Notes

1. An alternative programmable cycler, the "HYBrite Denaturation/Hybridization System," is produced by Vysis Inc. However, annealing and extension can be accomplished on thermoblocks or even in metal containers suspended in hot water baths (3). As always with PRINS, temperature is critical and must be carefully controlled.

2. In our experience, signal is increased by use of multiple (as many as four or five) primers for a single locus and by single-step annealing and extension.

3. Innis and Gelfand (8) note that at 20°C, TRIS buffer has a pKa of 8.3, and A pKa of -0.021/°C. Therefore, the actual pH of Tris-HCl may vary during thermal cycling.

4. An unamplified slide without TSAG reagents and an amplified slide without primer should be included as controls for each hybridization.

5. Slides should be kept moist during the PRINS procedure. If a humidified chamber is not available, cover slides with a damp paper towel in a closed box. If a humidifier is available, maintain humidity at 55-65% for optimal chromosome spreading (Subheading 3.3., step 10).

6. Treatment of slides with 0.02 N HCl removes loosely bound protein, thereby rendering DNA more accessible to the primers.

7. For each study, the primer concentration should be optimized. New England Nuclear recommends a 10-fold reduction in "probe" (primer) concentration as optimal. This step is a critically important one in PRINS because an improper concentration of probe can obviate development of the hybridization signal.

8. TaqStart monoclonal antibody binds Taq DNA polymerase, thereby minimizing nonspecific amplification and formation of primer dimers

9. Reagents should completely cover cells or metaphase spreads on slides.

10. After counting the A, C, G, and T nucleotide residues of the primers, compute annealing temperatures by use of either of the following formulas:

Tm = 69.3 + 0.41 (% G + C) - 650 / L where L = the length of the primer = the total number of nucleotides in the primer.

When different temperatures are obtained, the results may be averaged. In general, satisfactory annealing occurs at temperatures between 55 and 75°C. Higher temperatures increase annealing specificity.

11. Background staining is minimized by stringent washing of slides in SSC.

12. Low signal may be corrected by titration of HRP conjugate to optimize concentration, by increasing incubation time or concentration of amplification reagent, or by addition of a step to optimize penetration of reagents. Background staining may be minimized by decreasing concentration of HRP conjugate or primers, by increasing endogenous peroxide quenching, by filtration of buffers, or by increasing number of washes or the length of washes.

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