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.
Was this article helpful?