1. 100% Hank's (1): 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3. It is made from stock solutions that can be kept for several months. Sodium bicarbonate solution is made fresh. Solution 1: 8.0 g NaCl, 0.4 g KCl in 100 mL water. Solution 2: 0.358 g Na2HPO4, 0.6 g KH2PO4 in 100 mL water. Solution 3: 0.72 g CaCl2 in 50 mL water, Solution 4: 1.23 g MgSO47H2O in 50 mL water. Hank's premix: combine the following in order, 10 mL solution 1, 1 mL solution 2, 1 mL solution 3, 86 mL water, 1 mL solution 4. Store in refrigerator. Dissolve 0.35 g NaHCO3 in 10 mL water. Final 100% Hank's is 9.9 mL Hank's premix, 0.1 mL fresh sodium bicarbonate solution.
2. 10X Digoxigenin nucleotide labeling mix: 10 mM each ATP, GTP, CTP, 6.5 mM UTP, 3.5 mM digoxigenin-11-UTP. Store in aliquots at -20°C (available from Boehringer Mannheim #1277 073).
3. 10X Fluorescein nucleotide labeling mix: 10 mM each ATP, GTP, CTP, 6.5 mM UTP, 3.5 mM fluorescein-12-UTP. Store in aliquots at -20°C (Boehringer Mannheim #1685 619).
4. 10X TBE: dissolve 109 g Tris base, 55 g boric acid, 9.3 g diNaEDTA in 1 L of water. pH should be 8.3. 1X TBE is 90 mM Tris, 2.5 mM EDTA, 90 mM boric acid.
5. 10X Transcription buffer: 400 mM Tris-HCl pH 8.0, 60 mM MgCl2, 100 mM dithiothreitol, 20 mM spermidine, 100 mM NaCl, RNase inhibitor 1 U/|L. Store in aliquots at -20°C. (Supplied with RNA polymerase from Boehringer Mannheim.)
6. 20X SSC for hybridization and washing: (20X SSC is 3 M NaCl, 300 mM tri-sodium citrate). Dissolve 175.3 g NaCl and 88.2 g sodium citrate in 800 mL water. Adjust the pH with 1 M citric acid to 6.0. Adjust the volume to 1 L and sterilize by autoclaving.
7. Alkaline phosphatase (AP) inactivation buffer 0.1 M glycine-HCl, pH 2.2, 0.1% Tween-20.
8. Antidigoxigenin-AP Fab fragments (Boehringer Mannheim #1093274): 150 U/200 |L.
9. Antidigoxigenin-horseradish peroxidase (POD) Fab fragments (Boehringer Mannheim #1207733): 150 U lyophilized.
10. Antifluorescein-AP Fab fragments (Boehringer Mannheim #1426338): 150 U/200 |L.
11. Blocking solution for antibodies: 1X PBS, 0.1% Tween-20, 2 mg/mL BSA (BDH/ Merck #44155), 5% sheep serum (Gibco-BRL Life Technologies #035-6070H), 1% dimethylsufoxide, DMSO (Merck-BDH #28216).
12. DetectaGene™ Blue CMCG lacZ gene expression kit Molecular Probes D-2921.
13. DetectaGene™ Green CMFDG lacZ gene expression kit Molecular Probes D-2920.
14. DNase I, RNase-free (Boehringer Mannheim #776785).
15. Durcupan ACM (Sigma D0166).
16. ELF™ stop reaction buffer: 25 mM EDTA, 0.05% Triton X-100 in PBS, pH 7.2. Dissolve EDTA in PBS, and check pH.
17. ELF™-AP substrate kit (Molecular Probes Inc. Eugene #E-6601): dilute the substrate 1:100 in ELF™ reaction buffer supplied with the kit. Use within 30 min. Tissues should be equilibrated with pre-reaction wash buffer (30 mM Tris, 150 mM NaCl, pH 7.5) prior to adding the diluted substrate solution. The reaction is stopped with 25 mM EDTA, 0.05% Triton X-100 in PBS, and the final pH should be 7.2 (addition of 1.0 mM levamisole is optional, but is not necessary for zebrafish embryos).
18. Fast Red tablets, AP substrate (Boehringer Mannheim #1496549): each tablet contains 0.5 mg naphthol substrate, 2 mg Fast Red chromogen, and 0.4 mg levamisole. Store tablets for a short term at 4°C or at -20°C for long term. Wear gloves, and use plastic forceps to handle the tablets. Dissolve one tablet in 2 mL of 100 mM Tris-HCl, pH 8.2. Use the solution within 30 min.
19. Fluorescein di-P-d-galactopyranoside (FDG): Molecular Probes F-1179.
20. Glutaraldehyde: 50% glutaraldehyde for EM (BDH/Merck #36218) stock solution of glutaraldehyde is stored in aliquots at -20°C.
21. Gentamicin (Sigma G 1272) 10 mg/mL in water stock solution.
22. Hybridization mix: 50% formamide, 5X SSC, 0.1% Tween-20, 500 pg/mL yeast RNA (Sigma #R6750), 50 pg/mL heparin (Sigma #H9399), pH to 6.0, with 1 M citric acid.
23. Imagene Green™ C12FDG lacZ gene expression kit Molecular Probes I-2904.
24. Imagene Red™ C12RG lacZ gene expression kit Molecular Probes I-2906.
25. Paraformaldehyde/phosphate-buffered saline (PBS) fixative: paraformaldehyde is dissolved in PBS at 65°C. If it does not readily dissolve, add a drop or two of 1 M NaOH solution to pH 7.5. It should be cooled to 4°C and used within 2 d.
26. PBS: 130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4. For a 10X PBS, mix 75.97 g NaCl, 12.46 g Na2HPO42H2O, 4.80 g NaH2PO42H2O. Dissolve in less than 1 L of distilled water, adjust to pH 7.0 and to final volume of 1 L, sterilize by autoclaving.
28. Penicillin/streptomycin solution (Sigma P0781): 10,000 U penicillin and 10 mg/mL streptomycin in 0.9% sodium chloride.
29. Proteinase K (Boehringer Mannheim #1000144): make up a stock solution of proteinase K at 20 mg/mL in 50% glycerol 10 mM Tris, pH 7.8, and store at -20°C.
30. RNA polymerase SP6 (Boehringer Mannheim #810274).
31. RNA polymerase T3 (Boehringer Mannheim #1031163).
32. RNA polymerase T7 (Boehringer Mannheim #881767).
33. RNase inhibitor (Boehringer Mannheim #799017).
34. Sigma FAST™ Fast Red TR/Naphthol AS-MX (Sigma F 4648). Store at -20°C. Each tablet set contains the following when dissolved in 1 ml of water: 1.0 mg/mL
Fast Red TR, 0.4 mg/mL Naphthol AS-MX, 0.1 M levamisole, 0.15 mg/mL Tris buffer, pH 8.2. Remove required tablets from freezer, and allow to reach room temperature. Do not touch tablets with your fingers. Open Tris tablet (gold foil), and drop into a 1.5-mL microfuge tube. Add 1 mL of distilled water. Vortex until dissolved. Add Fast Red TR/Naphthol AS-MX tablet (silver foil) to Tris buffer. Vortex to dissolve. The solution should be used within 1 h. Reactions can be stopped by rinsing in PBS. Dispose of contaminated materials by incineration.
35. TE: 10 mM Tris HCl, 1 mM EDTA, pH 8.0. Make up with RNase-free water and autoclave.
36. Tricaine, 3-amino benzoic acid ethyl ester (Sigma A-5040) make a stock solution by dissolving 400 mg tricaine in 97.9 mL water with 2.1 mL 1 M Tris-HCl pH 7.0. Store in aliquots in freezer. Use 4.2 mL tricaine stock solution in 100 mL tank water.
37. True Blue™ peroxidase substrate (Kirkegaard & Perry Laboratories, #71-00-64).
38. Vector™ Red alkaline phosphatase substrate (Vector Labs SK-5100): Mix stock solutions as described with kit.
39. X-GAL, 5-bromo-4-chloro-3-indolyl-P-d-galactopyranoside, (Sigma B 4252): Dissolve in dimethylformamide at 20 mg/mL. Store at -20°C.
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