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

3.7.1. Simultaneous Immunological Detection of lacZ and Alkaline Phosphatase on Sections

This protocol allows for the staining of E. coli P-galactosidase antibody and NBT-BCIP histochemical detection of alkaline phosphatase. It is designed to detect alkaline phosphatase in primordial germ cells in lacZ-expressing embryos. All staining should be done at room temperature unless otherwise indicated and on 6-8 ^m sections.

1. Dissect out embryos in PBS medium and stain either the tail, head, yolk sac, or non-essential body part with X-gal staining solution to screen for transgenic embryos.

2. Fix embryos in 0.5% glutaraldehyde in PBS for 5-15 min, depending on the size of the embryos. As a general rule:

3. Wash embryos 2 x 10 min with PBS. The embryos then can be either stored in 70% ethanol for a short period of time, or dehydrated and embedded as described in Subheading 3.7.1., step 4.

4. Dehydrate embryos through 70, 80, 90, and 100% (x3) ethanol according to the times given below:

10-15 min 15 min 20-30 min

10 min 10-15 min 20-30 min

5. Transfer embryos to fresh polyester wax for 10-15 min. Repeat with fresh wax 2 x 10 min each. Embed with fresh polyester wax at room temperature, and section with either a cryostat or a microtome kept at 15°C (see Note 11).

6. Dewax sections in xylene (5 min), and rehydrate the sections by incubating them in a 37°C oven for 30 min followed by 2-min washes in 100% ethanol (2x at 37°C), 90, 70, 50, and 25% ethanol. Finally wash the sections for 5 min in dH2O.

7. Quench the endogenous peroxidase activity with 0.3% H2O2 in 40% methanol for 20 min, or 3% H2O2 in H2O for 5 min.

9. Block nonspecific binding by overlaying sections with 5-10% normal goat serum for 20 min.

10. Drain excess serum and incubate slides with E. coli ß-galactosidase antibody (diluted 1-5 |g/mL in PBS) for 60-75 min.

12. Incubate sections with biotinylated goat antirabbit IgG (2° Ab diluted 1:200 in PBS, 0.2% BSA, 3% normal goat serum) for 30 min.

13. Wash sections with PBS + 0.2%, BSA (3 x 10 min).

14. Incubate sections for 30 min with streptavidin peroxidase complex.

15. Wash sections with PBS + 0.2% BSA (3 x 10 min).

16. Incubate sections with peroxidase substrate solution (1 mg/mL DAB + 0.0225% H2O2) for 2-7 min or until desired staining intensity develops.

17. Wash sections for 5 min with dH2O.

18. Incubate sections with PBS (10 min).

19. Incubate sections with alkaline phosphatase buffer, pH 9.5 (2 x 10 min).

20. Incubate sections in NBT/BCIP substrate for 10-15 min in the dark at 37°C, or until desired intensity develops.

21. Wash the sections with dH2O (5 min).

22. Dehydrate and mount in Entellan.

3.7.2. Simultaneous Whole-Mount Histochemical Staining for ß-Galactosidase and Alkaline Phosphatase

1. Fix 7.5-11.5 d mouse embryos or older embryo (12.5-18.5 d) fragments and organs in 4% paraformaldehyde, 0.5% glutaraldehyde in PBS for 5-10 min.

2. Wash embryos in PBS (2 x 10 min). Incubate embryos for 10 min in alkaline phosphatase buffer.

3. Incubate embryos in NBT/BCIP substrate at 37°C for 10-20 min. Check intensity every 5 min.

4. Wash embryos extensively in X-gal washing buffer (staining solution without X-gal; 2 x 10 min).

5. Incubate embryos overnight in X-gal staining solution at 37°C.

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