Materials

2. Biotin-14-dATP, 0.4 mM stock solution (Life Technologies) or biotin-16-dUTP or other appropriate hapten-labeled nucleotide (see Note 2).

3. FITC-avidin D and FITC anti-avidin D antibodies (the latter is optional; Vector Laboratories) (see Note 3).

4. Oligolabeling buffer (OLB) for labeling with biotin-14-dATP: Prepare from the following stock solutions:

Solution A: 1.25 M Tris-HCl, pH 8.0, 125 mM MgCl2, 250 mM P-mercapto-ethanol, and 0.5 mM each dNTP (minus dATP; Roche). Solution A can be prepared as follows: To 485 |L of Solution O, add 9 |L of P-mercaptoethanol, 2.5 |L of 100 mM dCTP, 2.5 |L of 100 mM dGTP, and 2.5 |L of 100 mM dTTP. Store at -20°C. Do not add dATP to solution A (see Note 4). Solution B: 2 M HEPES, pH 6.6. Store at 4°C.

Solution C: Random hexamer pd(N)6 (Amersham Pharmacia Biotech) dissolved at 90 A260 units per milliliter in 1 mM Tris-HCl, 1 mM EDTA, pH 7.5. Store at -20°C. Solution O: 1.25 M Tris-HCl, pH 8.0, 125 mM MgCl2.

Mix solutions A, B and C in a ratio of 2 : 5 : 3 to give OLB. Store at -20°C.

5. Klenow fragment of DNA polymerase I (5 U/|L; Roche).

6. Reagents for ethanol precipitation to remove unincorporated nucleotides following the labeling reaction (see Note 5).

7. Vacuum filter glass funnel with fritted glass base and clamp (e.g., Fisherbrand, Millipore, or Kontes).

8. Nitex nylon mesh, approx 120-|im pore size.

9. Distilled water and distilled water containing 0.1% Tween-20 (v/v).

10. 50% Concentrated household bleach (e.g., Clorox) (i.e., bleach solution having a final concentration of approx 3% sodium hypochlorite.

11. Glass vial with snap cap (16 mL, Wheaton) or glass scintillation vial.

12. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 8 mM Na2PO4, 1.5 mM KH2PO4, pH 7.4. To make 1 L of 10X PBS stock solution, add 80 g NaCl, 2 g KCl, 2 g KH2PO4 and 11.4 g Na2PO4 to approx 800 mL of distilled water. Adjust to pH 7.4 with 10 N NaOH and add distilled water to 1 L.

14. Fixative: 3.7% Formaldehyde in PBS. Dilute 1 vol of 37% (w/w) formaldehyde (e.g., Sigma-Aldrich) in 9 vol of PBS. Formaldehyde is mutagenic and carcinogenic. Wear gloves and work with formaldehyde in a fume hood.

15. Methanol.

16. Heptane.

17. Prehybridization rinse solution: 4X SSC, 0.1% Tween-20, 20% formamide (e.g., Fisher Super Pure Grade). Deionize the formamide using ion-exchange resin AG501-X8 (BioRad). 20X SSC is 3 M NaCl, 0.3 M Na3-citrate, pH 7.0.

18. Hybridization solution: 4X SSC, 0.1% Tween-20, 50% formamide.

19. Wash solutions:

e. 2X SSC, 0.1% Tween-20. (This solution is also used prior to hybridization, at Subheading 3.3., step 7.)

20. Propidium iodide (PI) solution: 1 mg/mL PI in PBS. Store in the dark at 4°C. Spin at >10,000g for 5 min in a microcentrifuge before use. Other appropriate DNA stains can be used, depending on your microscope's light sources and filter sets (e.g., Sytox dyes and TOTO-3, from Molecular Probes).

21. 10 mg/mL RNase A. Required if using PI to stain DNA.

22. Mouse monoclonal antibody against Drosophila nuclear lamin Dm0 [e.g., ADL84; (25)] and a rhodamine-labeled secondary antibody (e.g, goat or donkey anti-mouse [Jackson ImmunoResearch] [optional]).

23. Fetal calf serum (FCS) (required for immuno-FISH). Store in aliquots at -20°C.

24. Microscope slides and cover slips.

25. Vectashield mounting medium (Vector Laboratories) or similar.

26. Nail polish, clear.

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