Materials

1. Forceps (e.g., Dumolux no. 5; Fine Science Tools).

2. Microscope slides and cover slips. Clean them in ethanol and keep them free of dust.

3. Glass scintillation vial with screw cap or similar glass vial (approx 10-16 mL).

4. Dust-off® (Falcon Safety Products, Inc., Sommerville, NJ) or similar compressed gas to blow dust from slides and cover slips.

5. Phosphate-buffered saline (PBS): 6 mM NaH2PO4, 6 mM Na2HPO4, 150 mM NaCl, pH 7.5. Autoclave or filter-sterilize (see Note 2).

6. PBST: PBS containing 0.1% (v/v) Tween-20 (polyoxyethylene-sorbitan mono-laurate, Sigma-Aldrich) (see Note 3).

7. Fixative: 10% (w/v) Formaldehyde, 1 mM EGTA in PBS, pH 7.5, freshly prepared. To make 10 mL of fixative, add 2.7 mL of 37% (w/w) formaldehyde solution, pH 7.5, and 100 ||L of 100 mM EGTA, pH 7.5, to 7.1 mL of PBS (see Notes 4 and 5).

8. Permeablization solution: PBS containing 0.3% (v/v) Tween-20.

9. Blocking solution: 10% Fetal calf serum (FCS) in PBST. To make approx 10 mL, add 1 mL of FCS to 9 mL PBST, filter-sterilize using a 0.2-|im syringe filter, and store at 4°C (see Note 6).

10. Primary and secondary antibodies (see Tables 1 and 2) (see Notes 7 and 8).

11. Propidium iodide (PI) solution: 1 mg/mL PI in PBS, or other DNA fluorochrome appropriate for your microscope.

12. 10 mg/mL RNase A (DNase-free): RNase A can be bought that is free of contaminating DNase activity. If in doubt, boil for 10-15 min to inactivate any DNases. Store in aliquots at -20°C. RNase A is required if staining DNA with PI.

13. Vectashield (Vector Laboratories) or similar antifade mounting medium.

14. Clear nail polish.

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