Primed In Situ Labeling Standard Protocol

1. Immerse slides in 0.02 NHCl for 20 min (see Note 6).

2. Immerse slides in 70% formamide-SSC, pH 7.0 for 2 min at 72°C, to denature chromosomal DNA.

3. Dehydrate slides by passage through a cold ethanol series, 70%, 85%, and 100% EtOH, 5 min each. Air-dry.

4. Prepare reaction mixture in a final volume of 40 pL containing 50 pmol of each oligonucleotide primer (see Note 7); 0.2 mMeach dATP, dCTP, dGTP; 0.02 mM dTTP; 0.02 mM biotin-16-dUTP; 50 mM KCl; 10 mM Tris-HCl, pH 9.0; 2 mM MgCl2; 0.01% BSA; and 1 unit Taq DNA polymerase with TaqStart antibody (see Note 8). Pipet 40 pL of reaction mixture onto freshly prepared slide (see Note 9).

5. Cover the working area of the slide completely with a glass cover slip. Attach the ends of the cover slip in place with a thin application of rubber cement.

6. Incubate slides. Our incubations are carried out on a programmable thermal cycler equipped with a flat plate for slides (MISHA, Shandon Lipshaw) (see Note 1). The program consists of one cycle of 10 min at annealing temperature (55-75°C) with an additional 30 min at 72°C for extension (see Note 10 for computation of annealing temperature).

7. After extension, slides are removed from cycler, the cover slips are removed, and the slides washed in 0.4X SSC at 72°C for 2 min to stop the reaction (see Note 11).

8. In our studies, biotin-labeled nucleotides are detected with the TSA Biotin System.

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