RNA In Situ Hybridization

1. Plasmid clone of cDNA with suitable RNA polymerase promoter sites (e.g., pBluescript, Stratagene).

2. 10X DIG RNA labeling mix (Roche): 10 mM ATP, 10 mM CTP, 10 mM GTP, 6.5 mM UTP, 3.5 mM DIG-UTP in Tris-HCl, pH 7.5.

3. RNA polymerase and buffer (for an antisense transcript, the one that transcribes from the 3' end of the gene) (New England Biolabs [NEB] or Roche).

4. RNAse-free water.

5. 2X Carbonate buffer: 60 mM Na2CO3, 40 mM NaHCO3, pH 10.2.

6. 2X Neutralization buffer: 200 mM sodium acetate, 1% (v/v) acetic acid.

2.4.2. Hybridization

1. Fix: 4% Paraformaldehyde in 100 mM HEPES, pH 6.9, 2 mM MgSO4, 1 mM EGTA. May be stored at -20°C. Check pH before (re)use.

3. Proteinase K (Roche). Stock is 19 mg/mL. Store at -20°C.

4. 2 mg/mL Glycine in PBST. Glycine stock is 200 mg/mL (store at room temperature).

5. Hybridization buffer (HB): 50% Formamide, 5X SSC, 100 |g/mL denatured sonicated salmon sperm DNA, 50 |g/mL heparin, 0.1% Tween-20, adjust to pH 4.5 with 2 M citric acid (approx 100 mM final concentration). Store at -20°C. Preheat for 65°C washes. 20X SSC is 3 M NaCl, 0.3 M sodium citrate, pH 7.0.

7. High pH buffer (HP): 100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween-20. Make up fresh, and add MgCl2 last to prevent precipitation.

8. Nitroblue tetrazolium (NBT): 18.75 mg/mL in 70% DMF stock (Roche).

9. X-Phosphate (5-bromo-4-chloro-3-indolyl-phosphate [BCIP]): stock solution is 50 mg/mL in DMF (Roche).

10. Alkaline phosphatase conjugated antidigoxygenin antibody (Roche), preadsorbed against embryos (see Note 1).

11. Gary's Magic Mountant (GMM): 1.6 g/mL Canada balsam (powder) in methyl salicylate. If only liquid Canada balsam is available, mix 4 : 1 with methyl salicylate.

12. 24-Well tissue culture plates and tissue culture inserts with 8-|im mesh (Falcon no. 3097).

13. Glass staining blocks.

14. Microscope slides and cover slips.

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