2. Paraformaldehyde (Sigma P6148). Prepare as a 4% solution in Ca2+/Mg2+-free PBS by heating to dissolve, but do not boil. Add two to three drops of 0.05 M NaOH to clear the solution (pH 7.5), which can then be stored at 4°C for 2 wk. Ideally, however, it should be freshly prepared. (Caution: Paraformaldehyde is toxic and readily absorbed through the skin. It is very destructive to the skin, mucous membranes, eyes, and upper respiratory tract. Therefore, glasses, gloves, and a mask should be worn to avoid contact with the dust and work should be performed in a fume hood.)
4. X-gal (4-chloro-5-bromo-3-indolyl-P-d-galactopyranoside; Progen, Richlands, QLD, 2000190). X-gal is prepared as a 4% (40 mg/mL) stock solution in dimethylformamide (DMF: Sigma D8654). Store at -20°C and protect from light.
5. Sorensen's phosphate buffer (PB), 0.1 M, pH 7.4: 0.02 M NaH2PO4 0.08 M Na2HPO4.
6. X-gal staining solution: 0.1% X-gal, 2 mM MgCl2, 5 mM EGTA, 0.01% (w/v) sodium deoxycholate, 0.02% (w/v) Nonidet P-40 (NP-40: Sigma), 5 mM K3Fe(CN)6 (potassium ferricyanide), and 5 mM K4Fe(CN)66H2O (potassium ferrocyanide) in 0.1 M pH 7.4, Sorensen's phosphate buffer. Potassium ferricyanide and potassium ferrocyanide can be made up as 500-mM stock solutions and should be stored at room temperature, protected from light for a maximum of 2 wk.
10. Paraffin wax.
11. Aluminum sulfate (Sigma, St. Louis, MO, A7523).
12. Nuclear fast red (0.1%; Merck, Rahway, NJ, 15939): Prepare by adding 500 mg of nuclear fast red and 25 g of aluminum sulfate to 500 mL of distilled water, heat/stir to dissolve, and filter when cooled.
13. Avertin: Prepare 100% stock solution by mixing 10 g of 2,2,2tribromethyl alcohol (Aldrich, Castle Hill, NSW, Australia, T4,840-2) with 10 mL of tert-amyl alcohol (Aldrich 24,048-6). To use, dilute 100% stock to 2.5% in water. Both the 100 and 2.5% stocks are stored wrapped in foil at 4°C. The correct dose of avertin will vary with each preparation. Therefore, several mice should be test injected within the range of 0.014-0.018 mL/g body wt.
15. Glycol methacrylate (2-hydroxyethyl methacrylate; Aldrich 12863-5).
16. Cresyl violet (Fluka, Buchs, Switzerland, 61120).
17. Acetic acid.
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