Visualizing lacZ by Immunostaining or Histochemistry in Combination with Alkaline Phosphatase Histochemistry

2. X-gal staining solution (see Subheading 2.4., item 6).

3. Glutaraldehyde (25%; Sigma G5882).

5. Polyester wax.

6. Xylene.

7. Hydrogen peroxide (H2O2).

8. Methanol.

9. Normal goat serum (Sigma G9023).

10. E. coli P-galactosidase antibody rabbit IgG (1° antibody; Cappel 55976).

11. Bovine serum albumin (Sigma Fraction V A9647).

12. Biotinylated goat antirabbit IgG (2° antibody; Immunodiagnostic PK6101): Store at 4°C.

13. Streptavidin-peroxidase complex (Vectastain Elite ABC reagent): Add two drops of reagent A to 5 mL PBS (with Ca2+ and Mg2+), mix well, then add two drops of reagent B, mixing immediately, and allow the ABC reagent to stand for about 30 min before use (Immunodiagnostic PK6101).

14. Diaminobenzedine (Dako S3000): Store at -20°C.

15. Peroxidase substrate solution: Dissolve one DAB tabletin 10 mL PBS to give 1 mg/mL DAB solution. Mix 2 mL of DAB solution with 15 mL of 3% H2O2 (or 1.5 mL of 30% H2O2). The substrate solution is stable for 2 h at room temperature. Unused stock DAB solution can be stored in the dark (wrapped in foil) for up to 5 d at 2-8°C, or longer at -20°C.

16. Alkaline phosphatase (ALP) buffer: 0.1 MTris-HCl, 0.1 MNaCl, 50 mMMgCl2, pH 9.5. Dissolve 12.1 L/g Tris-base and 5.84 g NaCl in dH2O, adjust pH to 9.5 with concentrated HCl, add 50 mL 1 M MgCl2, and make up volume to 1 L, (pH 9.5, alkaline phosphatase substrate package, Gibco-BRL, Gaithersburg, MD 8280SA).

17. NBT/BCIP substrate (Gibco-BRL 8280SA): Store at -20°C. Prepare NBT/BCIP substrate immediately prior to staining. Add 44 pL NBT to 10 mL alkaline phosphotase buffer, mix gently, then add 33 pL BCIP, mix gently.

18. Entellan (Merck).

19. 4% (w/v) Paraformaldehyde (see Subheading 2.4., item 2).

20. X-gal staining solution: 0.1% (w/v) X-gal, 2 mM MgCl2, 5 mM EGTA, 0.01% (w/v) sodium deoxycholate, 0.02% (w/v) NonidetP-40 (NP-40: Sigma), 5 mM

K3Fet(CN)6 (potassium ferriicyanide) and 5 M K4Fe(CN)66H2O (potassium fer-rocyanide) in 0.1 M pH 7.4 Sorenson's PB.

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