Whole-mount in situ hybridization of Hydra

Procedures

1. Anesthetize starved hydras with 1% urethane on ice. Hydras were starved more than 2 days before use.

2. Fix the hydras in 4% paraformaldehyde (PFA) overnight at 4ºC.

3. For bleaching, wash the hydras with 70% ethanol in 1×PBST (Phosphate-buffered saline, 0.1% Tween 20) 2×5 min.

4. Remove the ethanol solution and wash twice in 1×PBST.

5. To enhance the permeability, incubate the hydras in 10 ug/mL proteinase K solution at room temperature for 10 min. Glycine solution was added at the final concentration of 4 mg/mL to stop the reaction of proteinase K.

6. After 10 min, washed the hydras with PBST 2×5 min and refixed with 4% PFA at room temperature for 20 min.

7. Wash with PBST 2×5 min and incubate at 80ºC for 20 min to inactivate the activity of endogenous alkaline phosphatases.

8. Exchange the buffer into hybridization solution and incubate at 55ºC over 2 h.

9. Incubate the hydras with digoxigenin (DIG)–labeled RNA probes in hybridization solution at 55ºCovernight. DIG-labeled RNA probes were prepared by in vitro transcription with DIG RNA labeling mix (Rosch). Sense probes were used as the negative controls.

10. Gradually exchange the hybridization solution into 2×saline-sodium citrate (SSC) buffer + 0.1% CHAPS as described below:
Wash with 75% hybridization solution + 25% 2×SSC (5 min, 55ºC )

Wash with 50% hybridization solution + 50% 2×SSC (5 min, 55ºC )

Wash with 25% hybridization solution + 75% 2×SSC (5 min, 55ºC )

Wash twice with 2×SSC + 0.1% CHAPS (30 min, 55ºC) 

11. Wash the hybridized samples with 1×Maleic acid buffer (MAB) for 5min and then with 1% bovine serum albumin in MAB (MAB-B) for 1h.

12. Block the samples in 20% sheep serum in MAB-B at 4ºC over 2 h.

13. Incubate the samples in alkaline phosphatase–conjugated anti-DIG antibody (Rosch) (1:2000) overnight at 4ºC. anti-DIG antibody was preabsorbed before use.

14. Wash the samples in MAB 4×30 min.

15. Exchange the buffer into Alkaline phosphatase buffer (NTMT).

16. Add appropriate amount of nitro blue tetrazolium/bromochloroindolyl phosphate (NBT/BCIP) solution into NTMT and incubate under the dark condition.

17. After the coloring, wash the samples with NTMT and fixed with 4% PFA for 30 min.

18. Wash in 70% for 5 min and in 100% ethanol 2×10 min. Finally mount the sample with glycerin.

19. Observe the samples through Leica MZ10F (Leica) and capture the images using the digital camera D5200 (Nikon) equipped with the microscope.

Solutions

1. Components of hybridization solution

50% Formamide, 5×SSC, 200 ug/mL tRNA, 1×Denhardt’s, 100 ug/mL heparin, 0.1% Tween 20, 0.1% CHAPS in DEPC treated water.

2. Components of NTMT

100 mM NaCl, 100 mM Tris (pH 9.5), 50 mM MgCl₂, 0.1% Tween 20 in H₂O.