Primed In Situ Hybridization

1. Programmable thermal cycler equipped with a flat plate for holding slides (MISHA, Shandon Lipshaw, Pittsburgh, PA; see Note 1).

2. Dimethylsulfoxide (DMSO; Sigma Genosys, St. Louis, MO) molecular biology or high-performance liquid chromatography-grade.

3. Ethanol (Fischer Scientific).

4. Oligonucleotide primers (Research Genetics, Huntsville AL; see Note 2). The following probes for the SRY gene were high-performance liquid chromatogra-phy-purified, and stored at -20°C:


5. Trinucleotides (e.g., dATP, etc.): TSA kit; Tyramide Signal Amplification, for chromogenic and fluorescence in situ hybridization and immunohistochemistry (NEN Life Science Products, Boston, MA).

6. Biotin-16-2'-deoxyuridine 5'-triphosphate (dUTP; Roche Molecular Systems, Alameda, CA; formerly Boehringer-Mannheim).

8. Magnesium chloride (MgCl2) (Fischer Scientific).

9. Potassium chloride (KCl; Fisher Scientific).

10. Bovine serum albumin (Invitrogen Life Technologies, Carlsbad, CA).

11. Taq DNA polymerase (Amplitaq, Perkin-Elmer, Foster City, CA) with TaqStart antibody (Clontech Laboratories, Palo Alto, CA).

12. 20X SSC: 3 MNaCl, 0.3 MNa citrate, pH 7.0 (Invitrogen Life Technologies).

13. Formamide-SSC (Invitrogen Life Technologies).

14. TSA Biotin System. The kit should be kept at 4°C until used, although the blocking reagent may be kept at room temperature. With proper storage, the kit is useful for 6 mo, after which the contents may become unstable.

a. Prepare biotinyl tyramide stock solution: reconstitute biotinyl tyramide (amplifcation reagent) by addition of 0.3 to 1.2 mL of DMSO (the amount of DMSO will depend on the particular NEN kit used: 0.3 mL for 50-150 slides; 1.2 mL for 200-600 slides). Because DMSO freezes at 4°C, it is necessary to thaw the stock solution after removal from refrigerator.

b. Prepare TNT washing buffer: 0.1 MTris-HCl, pH 7.5, 0.15 MNaCl, 0.05% Tween-20. The manufacturer advises that PBS may be used as an alternative buffer and that 0.3% Triton X-100 may be used instead of 0.05% Tween-20.

c. Prepare TNB blocking buffer: 0.1 MTris-HCl, pH 7.5, 0.15 MNaCl, 0.5% Blocking Reagent (in kit). According to the manufacturer's protocol, the blocking reagent should be added slowly to buffer in small amounts while stirring. The mixture should be heated to 60°C with continuous stirring (see Note 3). The blocking reagent should be dissolved completely (this may take several hours). After preparation, store at -20°C.

15. Tween-20 (Sigma Genosys).

16. Triton X-100 (Sigma Genosys).

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