Protocol

Transcript In Situ Hybridization of Whole-Mount Embryos for Phenotype Analysis of RNAi-Treated Drosophila

This protocol was adapted from "RNAi in Drosophila" contributed by Richard W. Carthew, Chapter 17, in RNAi, A Guide to Gene Silencing (ed. Hannon). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

This protocol fixes and prepares embryos for in situ hybridization to visualize transcript expression patterns. It is a modification of the method developed by Tautz and Pfeifle for whole-mount in situ analysis of embryos. Use of the standard hybridization protocol on RNAi-treated embryos results in high background staining, which makes visualization of transcript expression patterns practically impossible. The following modifications eliminate this problem and allow visualization of transcript expression after RNAi injections.

MATERIALS

Reagents

Embryos (from Microinjection of dsRNA into Drosophila Embryos and Delivery of dsRNA into Drosophila Embryos by a Gene Gun)

Fixative A

Fixative B

Formamide/TE

Glycine block

Halocarbon oil

Purchase halocarbon oil Series 700, CAS# 9002-83-9 from Halocarbon Products Corporation, New Jersey.

Heptane

Hybe buffer

Hybe/PTW

Methanol (100%)

Methanol/PTW

Phosphate-buffered saline (PBS)

Proteinase K

Dissolve 10 µg/ml proteinase K in PTW buffer.

PTW buffer

Tape glue

Equipment

Coverslips

Double-stick tape

Egg strainer (Nitex mesh)

Eppendorf tubes

Forceps

Humidified chamber at 18-25ºC

Water baths preset to 48ºC and 75ºC

Kimwipes

Needle (21 gauge)

P1000 pipettor

Plastic dish (60 mm)

Spot dish (glass or porcelain)

Stereomicroscope

METHOD

1. Attach the embryos to coverslips with tape glue. Incubate the embryos under oil in a humidified chamber at 18-25ºC.

2. Collect the embryos at the appropriate stage. Use a razor blade to scrape excess oil from coverslip, taking care not remove embryos. Remove as much oil as possible.

3. Hold the coverslip over a 60-mm dish of heptane. Use a Pasteur pipette to wash the embryos on the slide with heptane until the oil is washed away (~6 sprays). Continue washing until all of the embryos are washed into the dish.

4. Transfer embryos to a well in the spot dish. At this point, the embryos are normally shrunken due to dehydration. Remove the heptane and incubate the embryos in 0.5 ml of H₂O (embryos float at the water surface). Allow them to rehydrate for 30 seconds to several minutes, monitoring under a stereomicroscope.

5. Replace the H₂O with Fixative A. Ensure that the well has both phases present. Cover with a slide and incubate for 20 minutes at room temperature.

6. Use a tip-cut P1000 pipettor to remove the embryos from the fix interface. Pipette the embryos onto the bottom of an upside-down egg strainer stuffed inside with Kimwipes. Let the solvent blot through the mesh leaving the embryos on top of the mesh.

7. Immediately pick up the embryos from the mesh with a strip of double-stick tape.

8. Flip the tape over and apply it to the bottom of a 60-mm plastic dish (embryo side up). Submerge tape and embryos under PBS.

9. Manually devitellinize the embryos with the tip of a 21-gauge needle and forceps. Popping the embryos with a nudge from the posterior is often sufficient to release them.

10. Remove the PBS from the dish and replace with PTW buffer. Pipette the embryos with PTW into an Eppendorf tube.

11. Remove PTW; replace with 1 ml of fresh PTW and incubate for 2 minutes.

12. Remove PTW and incubate in 1 ml of methanol/PTW (1:1) for 2 minutes.

13. Remove methanol/PTW and incubate in 1 ml of methanol for 2 minutes.

14. Remove methanol and incubate in 1 ml of methanol/PTW for 2 minutes.

15. Remove Methanol/PTW and incubate in 1 ml of PTW for 2 minutes.

16. Wash with PTW three more times.

17. Remove PTW and incubate in 1 ml of Fixative B for 20 minutes.

18. Remove Fixative B and wash embryos three times with PTW.

19. Remove PTW wash and digest embryos with proteinase K for 2 minutes.

20. Replace proteinase K solution with 1 ml of glycine block. Incubate for 10 minutes.

21. Remove glycine block and wash embryos twice with PTW.

22. Fix embryos a second time with Fixative B for 20 minutes.

23. Remove Fixative B and wash embryos five times with PTW.

24. Incubate embryos for 10 minutes in 1 ml of Hybe/PTW (1:1).

25. Replace with 100 µl of formamide/TE (1:1) and incubate for 10 minutes at 75ºC.

26. Plunge tube into ice-water slush to quick-chill the embryos. Allow to stand for 5 minutes.

27. Remove formamide/TE and incubate in 1 ml of Hybe buffer for 10 minutes.

28. Replace with a fresh aliquot of Hybe buffer and incubate for 60 minutes at 48ºC.

29. Hybridize embryos with an in situ probe at 48ºC as described by Tautz and Pfeifle. Perform washes, antibody reactions, and color reactions as described in Tautz and Pfeifle.

ACKNOWLEDGMENTS

I am thankful to several people who generously provided their time and help during the preparation of this chapter. Young Sik Lee contributed information and illustrations. Jason Kennerdell and Shinji Yamaguchi provided extensive input into the design of many of these protocols and provided unpublished results. Mike Nonet provided information and advice for construction and operation of his gene gun. I thank members of my laboratory for helpful comments on the manuscript.

REFERENCES

Tautz D. and Pfeifle C. 1989. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98: 81–85.[Medline]