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In situ Hybridization in Zebrafish Embryos   

Abstract

Materials and Reagents

1. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
2. PFA (powder) (Thermo Fisher Scientific)
3. DEPC (Sigma-Aldrich, catalog number: D5758 )
4. Glutaradehyde (Sigma-Aldrich, catalog number: G5882 )
5. Roche anti-DIG AP (Roche Diagnostics, catalog number: 11093274910 )
6. Torula yeast RNA (Sigma-Aldrich, catalog number: R6225 )
7. Heparin (Sigma-Aldrich, catalog number: H0777 )
8. Lamb/Sheep Serum (Thermo Fisher Scientific, catalog number: 16070-096 )
9. Formamide (High purity grade)
10. Methanol
11. Sodium Citrate
12. EDTA
13. NaCl
14. KCl
15. MgCl₂
16. Na₂HPO₄
17. KH₂PO₄ 
18. HCl
19. Tween 20
20. Citric acid
    
21. 1x PBT(made from DEPC H2O) (see Recipes)
22. Pronase (Roche Diagnostics, catalog number: 10165921001 ) (see Recipes)
23. NBT/BCIP color substrate (Promega Corporation, catalog number: S3771 ) (see Recipes)
24. Hybe⁺ buffer (see Recipes)
25. Hybe^- buffer (see Recipes)
26. 20x SSC (see Recipes)
27. 10x PBS (see Recipes)
28. 4% paraformaldehyde/PBS (see Recipes)
29. Blocking solution (see Recipes)
30. Stop solution (see Recipes)
31. Heat inactivated lamb serum (see Recipes)
32. Pre-staining buffer (see Recipes)
    

Equipment

1. Hybridization Incubator
2. 1.5 ml tube
3. 24-well plate
4. Aluminum foil
5. Nalgene filter
   

Procedure

1. Preparation of embryos
   1. Dechorinate the embryos using pronase. (Add 1 drop of pronase solution to 1 ml of E3). Keep the embryos at room temperature (RT) for 3-5 min (do not let the embryos in pronase solution for too long!).
   2. Pipet the embryos to break the chorion. 
   3. Collect the embryos in 1.5 ml tubes (usually 20 embryos/per tube). 
   4. Remove E3 buffer and rinse embryos with 1 ml of new E3 buffer to get rid of pronase.
      
   5. Remove all the E3 and add 500 μl of 4% PFA to fix the embryos O/N at 4 °C.
      
      
2. Dehydrate embryos
   1. Remove PFA (collect PFA in waste collecting tube) and wash with PBT three times (use PBT made from DEPC H₂O. 400 μl, 10 min each wash, total 3 times).
   2. Equilibrate with methanol to dehydrate the embryos (400 μl, 5 min each wash, three times).
   3. Remove methanol (collect methanol in waste collecting tube).
      
   4. Add 500 μl to each tube. Store the embryos in at -20 °C (at least 2 h).
      
      

1. Rehydrate embryos
   Wash for 5 min each sequentially in Methanol: PBT (3:1, 1:1, 1:3). (400 μl each wash, use DEPC-PBT.)
   Wash 4x, 5’ each in 100% PBT. (400 μl each wash)
   See note 1 in the end.
   Fix embryos in 4% PFA (+0.2% glutaradehyde) at RT for 20 min.
   Wash 4x, 5’ each in 100% PBT (400 μl each wash).
   
   
2. Hybridization
   Prehybridize embryos in hybe+ buffer (300 μl/tube) at 70 °C for 30 min-3 h.
   Replace prehybe with hybe+ buffer containing the probe(s) of choice. (0.1 ng-1 ng probe/μl hybe+ buffer)
   Incubate o/n at 70 °C.
   
   

1. Remove hybe/probe mixture and store at -20 °C. (can be used up to 3x)
2. Washes
   75% hybe-/25% 2x SSC         15min, 70 °C
   50% hybe-/50% 2x SSC         15min, 70 °C
   25% hybe-/75% 2x SSC         15min, 70 °C
   100% 2x SSC                         15min, 70 °C
   Wash 2 times in 0.2x SSC      30min, 70 °C
   75% 0.2x SSC/ 25% PBT       10min, RT
   50% 0.2x SSC/ 50% PBT       10min, RT
   25% 0.2x SSC/ 75% PBT       10min, RT
   PBT                                         10min, RT
   
3. Add blocking solution to block embryos in at RT for several hours (30 min minimum).
   
   

3. Antibody incubation
   1. After incubation, change buffer for antibody solution (1:5,000 dilution of Roche anti-DIG AP in blocking solution, 500 μl/tube), rock on a platform rotator, at 4 °C O/N.
      
      

1. Wash quickly in PBT. Use 500 μl/tube.
2. Wash 6x, 15 min in PBT, shaking at RT. 500 μl/tube.
3. Wash 1x, 5 min in pre-staining buffer (fresh made). 500 μl/tube. Transfer embryos to 24-well plate use plastic pipets.
4. Change the buffer to staining buffer+ (1 ml/well) to the embryos and wrap the plate with aluminum foil and shake at RT.
   
   1. Staining buffer+: Add 4.5 µl of NBT and 3.5 µl BCIP to 1 ml pre-staining buffer.
   2. Check new probes every 30 min to 1 h.
   3. When the staining is done, collect the staining buffer waste. Wash the stained embryos with 1 ml 2x PBT.
   4. Stop reaction by washing in stop solution.
   5. Leave the embryos in stop solution (1 ml/well) or fix in 4% PFA.
      
5. Store embryos in stop solution or 4% PFA at 4°C in a closed box.
   Note: For embryos older than 20 somite stage, permeabilization with proteinase K is required to allow the probe to enter the cells.
   
6. Incubate in Proteinase K (dilute 1 mg/ml stock 100 fold. 100 μl in 10 ml PBT).
   
   1. Late somitogenesis (14-22 sec): 2-4 min.
   2. 24 hpf: 10 min.
   3. 36/48 hpf: 20 min.
      
7. Wash once (quick) in PBT to get rid of the proteinase K (optional) and continue the fixation.

Recipes

1. Pronase
   30 mg/ml in E3
   
2. 20x SSC (pH7.0)
   NaCl              175.3 g
   NaCitrate       88.2 g
   for 1 L
   
3. 10x PBS
   To 800 ml ddH2O dissolve
   NaCl             80 g
   KCl               2 g
   Na2HPO4       14.4 g
   KH2PO4        2.4 g
   pH to 7.4 with HCl and add ddH₂O to 1 L.
   * Filter 1x PBS through a 0.2 μm Nalgene filter. Store at RT.
   
4. 1x PBT
   10x PBS (pH 7.4) to 1x PBS
   Make a 20% Tween stock. The final concentration of Tween for PBT should be 0.1%.
   
5. 4% Paraformaldehyde/PBS
   4 g in 100 ml of PBS, dissolve at 650 C, (or microwave while carefully watching)
6. Hybe⁺ buffer
   50% Formamide                                         25 ml of 100% stock
   5x SSC                                                      12.5 ml of 20x stock
   0.5 mg ml^-1 Torula yeast RNA                     1.25 ml of 20 mg/ml stock
   50 mg ml^-1 heparin                                     50 µl of 50 mg/ml stock
   0.1% Tween                                              250 µl of 20% Tween
   ddH₂O                                                       up to 50 ml
   50 ml total volume
   pH to 6-6.5 with 1 M citric acid ~460 µl.
   For Hybe-, leave out the torula yeast RNA and the heparin.
   
7. Heat inactivated Lamb Serum
   Thaw Lamb Serum and heat inactivate at 55 °C for 3 min. Store in aliquots at -20 °C.
   
8. Blocking solution
   100 mg BSA
   1 ml 100% Lamb/Sheep Serum
   50 ml PBT
   
9. 2x stop solution
   PBS (pH 5.5)
   EDTA 1 mM
   
10. Pre-staining buffer
    10 ml 1 M Tris (pH9.5)
    5 ml 1 M MgCl₂
    2 ml 5 M NaCl
    500 µl Tween 20
    to 100 ml with water.
    
11. Staining buffer ⁺
12. NBT/BCIP
    225 µl 50 mg/ml NBT
    175 µl 50 mg/ml BCIP
    to 50 ml w/ staining buffer
    

Acknowledgments

This protocol was modified from Reference 1, and developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA. This work was supported by NIH grant R01HD037975.

References

1. High-resolution in situ hybridization to whole-mount zebrafish embryos. Thisse C1, Thisse B. Nat Protoc. 2008: 3(1):59-69.