120807 yasuko

Protocol for Whole Mount In Situ Hybridization
https://www.brh2.jp

0. Prepare 100% EtOH
100% MetOH
50/50 Xylene/EtOH (1 ml/sample)
1. Transfer fixed embryos stored in 100% EtOH at -20 degrees to a 1.5 ml
tube with 100% EtOH
2. Rinse 100% EtOH, x5 (All rinses and washes are done in 1 ml, unlessotherwise mentioned.)
3. Rinse 50/50 Xylene/EtOH, x1
4. Rinse 100% EtOH, x5
5. Rinse 100% MetOH, x2
6. Prepare H2O2/MetOH (60 µl H2O2 + 840 µl MetOH; at least 450 µl/sample)
7. Incubate with rocking for 20 min in H2O2/MetOH.
During this incubation period, preparePBStw (1x PBS + 0.1% Tween 20)
Fixative (1.4 ml formalin + 8.6 ml PBStw)
50/50 MetOH/Fixative (1 ml/sample)
8. Rince 100% MetOH, x3
9. Incubate with rocking for 5 min in 50/50 MetOH/Fixative
Turn ON the Heat Block (55 degrees)
Prepare Ice Bucket
Take out Salmon Sperm DNA (ssDNA), Dextran, and Heparin fromthe freezer, and put them on ice after melting
10. Rinse PBStw, x5
11. Prepare ProK solution (1 ml PBStw + 4 µl Proteinase K (1 mg/ml); atleast 300 µl/sample)
12. Incubate for 3-4 min in ProK solution with occasional shaking
13. Rinse PBStw, x5
14. Post-fix in Fixative for 20-25 min with rocking
Boil ssDNA in a 1.5 ml tube for 5 min (12 µl/sample)
Prepare Hybridization solution (see next page) and50/50 Hybri(w)/PBStw (1 ml/sample)
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15. Rinse PBStw, x5
16. Incubate with rocking for more than 10 min in 50/50 Hybri(w)/PBStw
17. Rinse Hybri (w), x1
18. Pre-hybridize for at least 1.5 hr in 1 ml Hybri (h) at 55 degrees
19. Turn ON another Heat Block (80 degrees)
20. Prepare probe solution (just before use); add 0.5-1 µl RNA probe to 100 µl
Hybri (h), heat for 5 min at 80 degrees, and put immediately on ice.
21. Remove Pre-Hybridization solution
22. Add Probe solution, mix well, and let hybridize for more than 18 (12?) hrat 55 degrees.
Preparation of Hybridization Solutions, Hybri(w) and Hybri(h):a. Mix followings in a 50 ml tube:
Formamide

25 ml

20x SSC12.5 ml

50 mg/ml heparin

50 µl20% Tween20

250 µl

H2 O

2.2 ml40 mlb. Hybri (h): This solution is used for Pre-hybridization and Hybridization.
Transfer 4 ml of the solution (made at step a) into another tube.
Separate 880 µl of this solution into a 1.5 ml tube/sample.
Add 220 µl of 250 mg/ml Dextran/tube, mix well, and centrifuge.
Add 11 µl boiled ssDNA/tube, mix well, and centrifuge.
c. Hybri(w): This solution is used in washing steps.
Add 9 ml H2O into the remaining 36 ml solution (step a).
(Volume of water is 1/4 of the volume of the solution in step a.)(To prepare 50 ml Hybri(w), add 12.2 ml H2O instead of 2.2 ml at step a.)2120807 yasuko

23. Rinse Hybri (w), x1 at room temperature (RT)
24. Wash Hybri (w), for 10-15 min, x5 at 55 degrees
At each step, pre-warm Hybri (w) for the next step.
25. Wash 50/50 Hybri(w)/PBStw with rocking for 10 min, x1 (at RThereafter)
26. Wash 25/75 Hybri(w)/PBStw with rocking for 10 min, x1
27. Wash PBStw with rocking for 10 min, x5<<For Tyramide reaction, go to page 5>>
28. For AP staining, prepare an antibody solution by diluting the antibody
stock with PBStw.
Anti-DIG-AP (Roche), 1:1000
Anti-FITC-AP (DAKO), 1:10
Anti-DNP-AP (Vector), 1:50
29. Incubate in the antibody solution with rocking for more than 1.5 hr
30. Rinse PBStw, x1
31. Wash PBStw, for 5-10 min, x3
Prepare AP buffer (see next page)
32. Incubate in AP buffer, 5 min, x2
33. Transfer embryos into 24- or 4-dish.
34. Prepare Staining solution (see next page)
35. Incubate in Staining solution, protect from light.
Examine the coloring reaction at some intervals, without turning on light.
36. When good staining is observed, stop the reaction by rinse with PBStw
several times.
37. Post-fix for more than 20 min at RT or over night at 4 degrees.
38. Rinse PBStw, x3-5
39. Incubate in AP inactivation solution with rocking for more than onehour.
40. Rinse PBStw, x3-5
41. For samples stained with NBT/BCIP, EtOH treatment: Incubate in 50/50
PBStw/EtOH for 5 min; 100% EtOH 5-10 min, x1 or 2; 50/50 PBStw/EtOHfor 5 min; Rinse PBStw, x3-5
42. Detection of another probe (go to step 28), Detection of Biotin-dextran,3120807 yasuko

Counter-stain with DAPI, or Observation.
Preparation of AP buffer and Staining solution<Staining with NBT/BCIP>
AP buffer
5M NaCl

400 µl1M MgCl2

1 ml

2M Tris-Cl (pH9.5) 1 ml
20% Tween20
H2 O

100 µl17.5 ml
20 ml

Staining solution
Mix followings in 1ml AP buffer and centrifuge At least 500 µl/sample.
50 mg/ml NBT 6.6 µl
50 mg/ml BCIP 3.3 µl

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Tyramide Reaction
Samples from Step 27 (page 3)
1. Rinse with TNT (0.1 M Tris (pH7.5), 0.15 M NaCl, 0.05% Tween20).
1 M Tris, 5 ml; 5 M NaCl, 1.5 ml; 20% Tween20, 125 µl/50 ml
2. Blocking: Incubate with rocking in Blocking solution (PE) for more than 30
min at RT.
3. Dilute an Antibody stock solution with Blocking solution
Anti-DNP-HRP (PE), 1:100
4. Incubate with rocking in Antibody solution for more than 1.5 hr at RT
5. Rinse TNT, x1
6. Wash TNT, for 4 min x3
Prepare 4-Iodophenol: 30 mg 4-Iodophenol dissolve in 1 ml of 100%
EtOH
Prepare H2O2: add 1 µl H2O2 in 100 µl H2OTake out 250 mg/ml Dextran from freezer
7. Rinse Borate Buffer (pH8.5), 4min x2
Borate, 0.309 g; 5M NaOH, approx. 100 µl; 20% Tween20, 250 µl/50 ml
8. Prepare Tyramide Reaction solution:
H2O2 (1/100)

1 µl

4-Iodophenol (30 mg/ml)

1 µl

Dextran (250 mg/ml)

8 µl

Borate Buffer 100 µl ßMix well, centrifuge, and add appropriate Tyramide.
1 µl for DNP (DNP amplification reagent)Mix well and centrifuge
9. Incubate in Tyramide Reaction solution for 15 min.
10. Rinse with PBStw x2
11. Wash PBStw for 4 min x3

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120807 yasuko<Staining with Vector Red>
1. Dilute an Antibody stock solution with Blocking solution
Anti-DNP-AP (Vector), 1:100
2. Incubate with rocking in Antibody solution for 1.0 hr at RT
3. Rinse PBStw x1
4. Wash PBStw for 5min x 3
6. Wash APtw (pH8.5) buffer 5min x2
7. Rinse AP (pH8.5) buffer x1 (not entirely)
8. Incubate in Staining solution for (protect from light).
Examine the coloring reaction at some intervals. If the signal is notstrong at 45 min, the staining solution may be replaced with a new one.
9. Stop the reaction by rinsing with PBStw several times.
10. Post-fix for more than 20 min at RT or over night at 4 degrees.
11. Rinse with PBStw, x3-5
12. Incubate in AP inactivation solution with rocking for more than one hour.
13. Rinse PBStw, x3-5
AP buffer
5M NaCl

400 µl1M MgCl2

1 ml

2M Tris-Cl (pH8.5) 1 ml
H2 O

17.5 ml
20 ml

Separate an appropriate volume (2.5-10 ml) of this buffer.
Add 20% Tween20 in the remaining AP buffer for final 0.1%.
Staining solution
Add 1 drop of sol. 1 (supplied in a Vector Red kit), 1drop of sol. 2, and 1 dropof sol. 3 into 2.5 ml AP buffer without Tween20.
Change to fresh Vector Red Staining solution every 45 min.
*NBT/BCIP should be used first, and Vector Red is used second.
**Signal can be amplified by combining a-DNP-HRP + DNP-Tyramide anda-DNP-AP.
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