Preparation of Material for Sectioning

1. Diethylpyrocarbonate (DEPC) treated water. DEPC (Sigma, Poole, UK) is added to double-distilled water to a concentration of 0.1% (v/v). The solutions are shaken and then autoclaved. All solutions used for steps between sectioning tissue and the first post hybridization wash are prepared in DEPC water. All subsequent steps use solutions prepared in double-distilled water

2. 10X PBS: 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.

3. 4% (w/v) paraformaldehyde in PBS freshly prepared on the day of use for pretreat-ment of sections, but can be stored for fixation of embryos. Sixteen grams of paraformaldehyde (Fluka no. 76240, Gillingham, UK) are weighed out in a fume hood—wear mask and gloves. This is added to 400 mL of 1X PBS in distilled water and heated at 65°C in a water bath 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. 10X Saline: 8.3% (w/v) NaCl. Add DEPC to 0.1% and autoclave.

5. Howard's Ringer (0.12 M NaCl, 0.0015 M CaCl2, 0.005 M KC1. Per liter: 7.2 g NaCl, 0.17 g CaC12, 0.37 g KC1, pH 7.2, with very dilute HC1).

6. Absolute (100%) ethanol and graded ethanols 70, 80, and 95% (v/v) prepared with DEPC water.

8. Histoclear (National Diagnostics, Hull, UK).

10. Plastic moulds for wax (e.g., R.A.Lamb, London, UK).

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