Visualizing lacZ by Histochemistry in Combination with In Situ Hybridization

1. PBS (without Ca2+- and Mg2+) (see Subheading 2.4., item 2).

2. 4% Paraformaldehyde (Sigma: see Subheading 2.4., item 2 for preparation).

4. Xylene.

5. Paraffin wax.

6. TESPA (3-aminopropyltriethoxy-saline; Sigma A3648): TESPA coated slides are prepared as follows. Wash slides in 10% HCl/70% ethanol, followed by 95% ethanol for 1 min each, and then air-dry. Dip the slides in 2% TESPA in acetone for 10 s. Wash twice with acetone and then with distilled water before drying at 37°C.

7. DIG RNA labeling kit (Boehringer Mannheim 1175025).

12. Hybridization buffer: 5X SSC, 50% (v/v) deionized formamide, 0.4% (w/v) SDS, 0.1% (w/v) N-lauroylsarcosine, 2% (w/v) blocking reagent (Boehringer Mannheim 10961761).

17. Normal sheep serum (Sigma S3771).

18. DIG antibody-sheep conjugated alkaline phosphatase (dilute 1:200 in 150 mM NaCl/100 mM maleic acid/10% sheep serum; Boehringer Mannheim 1093274).

19. Alkaline phosphatase buffer (ALP; see Subheading 2.6., item 16).

20. NBT/BCIP substrate (see Subheading 2.6., item 17).

21. E. coli P-galactosidase antibody rabbit IgG (5 |g/mL in PBS/20% normal goat serum; Cappel 55976).

22. Normal goat serum (Sigma G9023).

23. Biotinylated goat anti-rabbit IgG (2° antibody; diluted 1:200 in PBS/0.2%BSA; Immunodiagnostic PK6101) for 60 min at room temperature.

24. Streptavidin-P-galactosidase conjugate (diluted 1:200 in PBS/0.2% BSA; Boehringer Mannheim 1112481).

25. X-gal washing buffer (X-gal staining solution without X-gal; see Subheading 2.4., item 6).

26. X-gal staining solution (see Subheading 2.6., item 20).

27. CHAPS (3-[{3-cholamidopropyl} dimethylammonio]-1-propansulfonat; Boehringer Mannheim 810118).

30. Hydrogen peroxide (H2O2).

31. Dimethylsulfoxide (DMSO; Sigma D2650).

32. Glycine.

33. Glutaraldehyde (25% w/v; Sigma G5882).

35. Prehybridization solution consists of the following and should be prepared on the day of use: 50% deionized formamide, 5X SSC, 2% blocking powder, 0.1% Tween-20, 0.5% CHAPS, 50 pg/mL yeast RNA, 5 mM EDTA, 50 pg/mL heparin.

36. Bovine serum albumin (Fraction V; Sigma A9467).

37. Mouse embryo powder: This is prepared by homogenizing 12.5-14.5-d mouse embryos in a minimum vol of PBS. Add 4 vol of ice-cold acetone, mix, and incubate on ice for 30 min. Spin at 10,000g for 10 min and remove the supernatant. Wash the pellet with ice-cold acetone, and spin again. Spread the pellet out, and grind into a fine powder on a sheet of filter paper. Allow the powder to air-dry, and store in an air-tight tube at 4°C.

38. NTMT: 100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween-20.

39. Levamisole (2 mM in NTMT; Sigma L9756).

40. Color reaction mix consists of the following and should be prepared immediately before use: 4.5 mL NBT (Gibco-BRL 8280SA), 3.5 pL BCIP (Gibco-BRL 8280SA) in 1.0 mL NTMT.

Was this article helpful?

0 0

Post a comment