Fixation Processing and Hybridization

All solutions are prepared in DEPC-treated water.

1. Phosphate-buffered saline (PBS; 10X concentrated): 1.3 M NaCl, 70 mM Na2HPO4, 30 mM NaH2PO4. Check that pH is 7.0 (if required, adjust slightly with HCl or NaOH as appropriate). Add DEPC to 0.1% and autoclave.

2. Howard's Ringer: 0.12 M NaCl, 0.0015 M CaCl2, 0.005 M KCl. Per liter: 7.2 g NaCl, 0.17 g CaCl2, 0.37 g KCl, pH 7.2, with very dilute HCl.

3. Paraformaldehyde: 16 g of paraformaldehyde (Fluka, Gillingham, UK, no. 76240) is weighed out in a fume hood wearing mask and gloves. It is added to 400 mL of 1X PBS in distilled water and heated at 65°C in a water bath in the fume hood with occasional swirling until dissolved (about 90 min). It is cooled to 4°C prior to use or stored in 20 mL aliquots at -20°C.

4. PBT is 1X PBS containing 0.1% (v/v) Tween 20 (Sigma).

5. Methanol (AnalaR; Merck BDH).

6. Hydrogen peroxide (30% w/w; Sigma, replace every 4 mo).

7. Proteinase K (Boehringer Mannheim, Lewes, UK) 20 mg/mL and stored in single-use (15-20 |L) aliquots at -20°C.

8. 25% (w/v) Glutaraldehyde (electron microscopy grade; BDH, Poole, UK). Glut-araldehyde is carcinogenic and should be used in a fume cupboard.

9. Prehybridization/hybridization solution: 50% (v/v) deionized formamide (Fisons, Loughborogh, UK), 5X SSC, pH 4.5 (20X SSC stock is 3 MNaCl, 0.3 M sodium citrate; use citric acid to pH), 50 mg/mL yeast RNA (Sigma), 1% (w/v) sodium dodecyl sulfate (SDS), 50 mg/mL heparin (Sigma). Heparin and yeast RNA are stored as concentrated 50 mg/mL solutions at -20°C.

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