Whole Mount In Situ Protocol
(Wilkinson/Vogt
lab) revised C. Thut 2/27/01 version 1.0
1. Dissect embryos in PBS, removing extra-embryonic membranes and opening cavities that may trap reagents.
2. Fix in 4% paraformaldehyde in PBS, 2 hrs to O/N at 4C. (For eyes, 30 min- 2 hrs).
3. Wash 2X PBT, 5 min., 4C.
4. Wash 5 min, 4C in 25% methanol (in PBT)
5. Wash 5 min, 4C in 50% methanol.
6. Wash 5 min, 4C in 75% methanol.
7. Wash 2X for 5 min, 4C in 100% methanol. Can store at 20C.
8. Rehydrate embryos through this methanol/PBT series in reverse, then wash 2X with PBT.
9. Bleach with 6% hydrogen peroxide in PBT for 1 hour.
10. Wash 3X in PBT, 5 min.
11. Treat with 10 ug/ml proteinase K in PBT. (12.5 min for E8.5, 15 min for E10.5, 20 min for E13.5 and 18 min for embryos stages in between). For dissected eyes, use 0.2 0.5 ug/ml proteinase K for 5 10 minutes.
12. Wash 10 min with fresh 2 mg/ml glycine in PBT.
13. Wash 2X 5 min with PBT.
14. Refix embryos with 0.2% glutaraldehyde/4% paraformaldehyde in PBT for 20 min. (Made from 25% glutaraldehyde stock from Sigma.)
15. Wash 2X, 5 min PBT.
16. Add 1 ml pre-hybridization mix and transfer the embryos to a 1.5 ml microtube.
17. Replace 1 ml pre-hyb mix and incubate at 70C, 2 hr. (Embryos can be stored indefinitely in this mix at 20C).
18. Add 0.4ml hybridization mix including approx. 1 ug/ml digoxigenin-labelled RNA probe.
19. Incubate at 70C, O/N with rocking.
PBS, 0.1% Tween-20
50% formamide 5 mls (100%)
5X SSC pH 5.0 2.5 mls (20X)
50 ug.ml yeast RNA 10 ul (50 mg/ml)
1% SDS 1 ml (10%)
50 ug/ml heparin 10 ul (50 mg/ml)
DEPC water 1.48 mls
20. Wash the embryos 3X with solution 1 (50% formamide, 5XSSC pH 5, 1% SDS) for 30 min at 70C.
21. Wash 3X with solution 3 (50% formamide, 2X SSC pH 5) for 30 min at 65C.
22. Wash 3X for 5 min with TBST.
23. Pre-block the embryos by incubating with 10% heat-inactivated sheep serum in TBST for 2.5 hrs.
24. During this, pre-absorb the antibody as follows:
a) Add a small amount of embryo powder into a microtube, add 1.5 ml TBST and nutate at 70C for 30 min, then vortex for 10 min.
b) Cool on ice and add 15 ul sheep serum and 1.5 ul antidigoxigenin antibody coupled to alkaline phosphatase (Boehringer).
c) Shake gently at 4C for 1 hour, then spin at 4C for 10 min. Recover the supernatant and dilute it to 2 ml with 1% sheep serum in TBST.
25. Remove the 10% serum from the embryos, rinse with 0.5 Ab solution then replace with 1 ml preabsorbed antibody and rock overnight at 4C.
1.4 M NaCl
27 mM KCl
0.25 M Tris-HCl, Ph 7.5
1% Tween-20
1. Homogenize 12.5 14.5d mouse embryos in a minimum volume of ice-cold PBS.
2. Add 4 vol ice-cold acetone, mix, and incubate on ice for 30 min.
3. Centrifuge at 10000g for 10 min, remove the supernatant, and then wash the pellet with ice-cold acetone and spin again.
4. Spread the pellet out and grind it into a fine powder ona sheet of filter paper.
5. Air-dry the powder and store at 4C.
Day 3--Washes and histochemistry
26. Wash the embryos at RT 3X for 5 min each with TBST.
27. Wash 5X at RT for 1 1.5 hours each with TBST.
28.
At this point, the Vogt lab washes overight at 4C in
TBST, but I have skipped this step for embryonic eyes and itıs looked
fine. Should probably do it if
youıre using whole embryos.
29. Wash 3X for 10 min each with NTMT.
30. Incubate the embryos with NTMT plus NBT and BCIP (for 50 ml NTMT add 405 ul NBT and 210 ul BCIP).
31. Cover reactions with foil and nutate at RT. Check often, staining can occur in as little as 5 minutes.
32. When judged complete, wash 2X in PBT pH5.5 for 10 min at RT.
33. Postfix with 4% paraformaldehyde, 0.1% glutaraldehyde in PBS for 1 hr at RT.
34. To clear, wash in increasing concentrations of glycerol, 30%, 50%, 70%, 80% for 15 min 1 hr, depending on embryo size.
Solutions:
100 mM NaCl 1 ml 5M NaCl
100 mM Tris HCl, pH 9.5 5 ml 1M Tris HCl, pH 9.5
50 mM MgCl2 2.5 ml 1M MgCl2
0.1% Tween-20 0.5 ml 10% Tween-20
41
ml H2O
50 ml