3.8.1. Simultaneous Detection of Oct 4 mRNA and ß-Galactosidase Protein on Paraffin Sections
1. Fix 7.5-11.5 d embryos or fragments and organs of older embryos (12.5-18.5 d) in 4% paraformaldehyde overnight at 4°C, and then wash in PBS (2 x 10 min).
Dehydrate the embryos according to the schedule given in Subheading 3.7., step 4, wash in xylene (3 x 10 min), followed by paraffin wax (3 x 15 min), and then embed the embryos in fresh paraffin wax before storing at 4°C. Cut sections at 8 mm, and mount on TESPA-coated slides.
2. A 600 bp Oct4 RNA digoxigenin probe was generated by in vitro transcription with T3 (antisense strand) and T7 (sense strand) RNA polymerase (DIG RNA labeling kit, Boehringer Mannheim, cat no. 1175025).
3. Dewax the embryos in xylene (5 min), rehydrate (100% x 2, 90, 80, 70, 50, dH2O; for 1 min each), and then wash the sections in PBS (2 min).
4. Refix the sections in 4% paraformaldehyde (10 min), and then wash with PBS (2 x 5 min).
5. Treat the sections with 0.3% Triton X-100 in PBS (15 min), and then wash in PBS (2 x 5 min).
6. Incubate the sections in 37°C prewarmed proteinase K (20 mg/mL; 5 min), wash in PBS (3 x 10 min), and then post-fix the sections in 4% paraformaldehyde (15 min).
7. Prehybridize the sections for 3 h in hybridization buffer (5X SSC, 50% delonized formamide, 0.4% SDS, 0.1% N-lauroylsarcosine, 2% blocking reagent.
8. Dilute the probe in hybridization buffer containing 50 pg/mL heparin and 20 mg/mL yeast tRNA to a final concentration of 0.05 ng/mL. Heat the probe at 80°C (10 min) and immediately store on ice until addition to the slides. Incubated with sections with 500 pL of probe at 55°C in a humidified chamber overnight.
9. Wash the sections at room temperature in 2X SSC/0.2% SDS (2 x 10 min); then at 58°C in 0.5X SSC/0.2% SDS (2 x 30 min) followed by 150 mM NaCl/100 mM maleic acid (pH 7.5; 10 min).
10. Block the section in 150 mM NaCl/100 mM maleic acid (pH 7.5) containing 10% sheep serum (30 min), and then incubate with diluted alkaline phosphatase-con-jugated sheep anti-DIG antibody (1:200 dilution) for 2 h.
11. Wash the sections in 150 mM NaCl/100 mM maleic acid (pH 7.5; 2 x 15 min), then in alkaline phosphatase buffer (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5, 10 min), and incubate with NBT/BCIP substrate. AlIow the color to develop in the dark for 60-90 min at room temperature.
12. Wash the sections in PBS (3 x 10 min), block with 20% normal goat serum in PBS (30 min), and then incubate with 5 pg/mL E. coli anti-P-galactosidase antibody overnight at 4°C.
13. Wash the sections in PBS containing 0.2% BSA (3 x 10 min) and then incubate with biotinylated goat anti-rabbit IgG (2° antibody; diluted 1:200 in PBS/ 0.2%BSA) for 60 min at room temperature.
14. Wash the sections in PBS containing 0.2% BSA (3 x 10 min), and then incubate with streptavidin-P-galactosidase conjugate for 60 min (1:200 dilution).
15. Wash the sections in PBS (2 x 10 min), then in X-gal washing buffer (10 min), and the incubate in X-gal solution at 37°C (20 min) in the dark.
16. Dehydrate the sections, and mount in Entellan.
3.8.2. Whole-Mount In Situ Hybridization Combined with X-Gal Staining
All steps are performed at room temperature unless otherwise stated.
1. Fix embryos in 4% paraformaldehyde for 5 min.
2. Stain embryos in X-gal staining solution for 1 h.
3. Refix embryos in 4% paraformaldehyde overnight.
5. Wash embryos in 25, 50, and 75% methanol/PBT (5 min), and then in 100% methanol (2 x 10 min). Embryos can be stored in 100% methanol at -20°C.
6. Fix the embryos in methanol:DMSO (4:1) for 1 h and then bleach the embryos in methanol:DMS0:30%H202 (4:1:1) for 1 h.
7. Rehydrate embryos in 75, 50, and 25% methanol/PBT (5 min), and then wash the embryos in PBT (2 x 5 min). It is important to poke a few holes with a small needle into these embryos prior to washing to facilitate the flow of solutions.
8. Treat the embryos with Proteinase K (6 mg/mL; 5 min).
9. Wash the embryos in 0.2% glycine in PBT (2 x 5 min).
10. Refix the embryos in 4% paraformaldehyde/0.2% glutaraldehyde in PBS (30 min).
12. Wash embryos in 1 mL of prehybridization solution.
13. Prehybridize embryos in 1 mL of prehybridisation solution at 65°C (3 h). Embryos can be stored at this point at -20°C in prehybridization solution.
14. Incubate embryos in 1 mL of hybridization solution at 60°C overnight.
15. Posthybridize wash with each of the following for 5 min at 60°C:
a. 100% Prehybridization solution;
b. 75% Prehybridization solution: 25% 2X SSC;
c. 50% Prehybridization solution: 50% 2X SSC;
d. 25% Prehybridization solution: 75% 2X SSC.
16. Wash with each of the following for 30 min at 60°C:
18. Preblock embryos in 10% normal sheep serum/1% BSA in PBT at 4°C (3 h).
19. Pre-absorb the antibody at 4°C (3 h) with 3 mg mouse embryo powder, 500 |L of 10% normal sheep serum, 1% BSA in PBT, and 1 |L of anti-DIG antibody, rocking the mixture gently. Spin the solution at 2000g for 5 min and remove the supernatant (preabsorbed antibody).
20. Incubate the embryos in preabsorbed antibody overnight at 4°C with constant rocking.
22. Wash embryos in 2 mM levamisole in NTMT (2 x 10 min).
23. Wash embryos in NTMT (10 min).
24. Incubate embryos in color reaction mixture until sufficient coloration is visible in the specimen.
25. Refix embryos in 4% paraformaldehyde overnight, wash in PBS and examine (see Notes 12 and 13).
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