Embryonic In Situ Hybridization for the Tardigrade Hypsibius exemplaris

Embryo Collection

• 1. In a 35 mm × 10 mm Petri dish filled with spring water, slice through the middle of maternal exuviae filled with embryos with a 25-gauge needle attached to a sterile 1-mL syringe (see Supplemental Movie S1).

• 2. Transfer embryos to a 1.5-mL tube filled with 0.5× PBTw.

  • We routinely follow this protocol with 20–50 embryos per 1.5-mL tube.

Primary Permeabilization

• 3. Centrifuge the 1.5-mL tube containing embryos at 18,500g for 3 min. Remove most of the liquid, leaving embryos suspended in ∼ 20 µL of 0.5× PBTw.

• 4. Add 20 µL of chymotrypsin/chitinase solution, and let stand for 1 h. Centrifuge at 3000g for 3 min, and remove most of the liquid.

• 5. Wash three times as follows: Add 500 µL of 0.5× PBTw, let stand for 5 min, centrifuge at 3000g for 3 min, and remove most of the liquid.

Paraformaldehyde Fixation

• 6. Wash with 1 mL of fixative solution while shaking vigorously on a VWR Genie 2 G560 Vortexer set to shake speed 3 for 30 min. Centrifuge at 3000g for 3 min, and remove most of the liquid.

• 7. Wash five times as follows: Add 500 µL of 0.5× PBTw, let stand for 5 min, centrifuge at 3000g for 3 min, and remove most of the liquid.

Cleaning Mobicol Mini-Columns

• 8. Set up mini-columns as in Figure 1A and B, and then wash them four times as follows:

  • i. Unscrew the Leur-lock cap, add 600 µL of 0.5× PBTw to the top of the mini-column, and then screw the Leur-lock cap and syringe back on.

  • ii. Push the syringe plunger all the way down, and discard the liquid that collects in the tube.

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Figure 1.

(A) The Mobicol mini-column configuration used in this protocol. The black dashed line denotes the approximate level to which we reduce the embryo suspension during wash steps. (B) The mini-columns are kept in a tube rack for this protocol. (C) The mini-column configuration while the embryos are stored in methanol at −20°C.

Methanol Dehydration

• For all mini-column washes below, unscrew the Leur-lock cap, add the liquid to be used for washing the embryos, and then screw the Leur-lock cap and syringe back on. After letting the embryos stand in the liquid for the specified time, push the liquid through the mini-column with a 1-mL syringe (see the black dashed line in Fig. 1A). The embryos must remain submerged in liquid at all times, so during these washes, pull back on the syringe plunger to form a vacuum in the mini-column to ensure that not all of the liquid is lost.

• 9. Transfer the embryos to a clean mini-column using a 9 inch glass Pasteur pipette.

• 10. Wash embryos for 5 min in 500 µL of 25% methanol in 0.5× PBTw.

• 11. Repeat Step 10 with 50%, 70%, and 90% methanol in 0.5× PBTw.

• 12. Quickly wash the embryos three times in 500 µL of 100% methanol. After adding the final wash of 100% methanol, insert a bottom plug into the column and replace the Luer-lock cap with a closing cap (Fig. 1C). Leave the tube at −20°C for at least 20 min.

Methanol Rehydration

• 13. Remove the bottom plug, and replace the closing cap with the Luer-lock cap and syringe. Wash the embryos for 5 min each in 500 µL of 90%, 70%, 50%, and 25% methanol in 0.5× PBTw, followed by three quick washes with 500 µL 0.5× PBTw.

Secondary Permeabilization

• 14. Use a 9 inch glass Pasteur pipette to transfer the embryos from the mini-column into a 35-mm dish filled halfway with 0.5× PBTw.

• 15. Cut the embryos out of the eggshells with a 25-gauge needle attached to a 1-mL syringe (see Supplemental Movie S2).

• 16. Re-collect the embryos in a clean mini-column in 500 µL of 0.5× PBTw, and then push the excess liquid through the mini-column with a 1-mL syringe.

Acetylation

• 17. Quickly wash the embryos with 500 µL of 0.5× PBTw.

• 18. Quickly wash the embryos twice with 500 µL of 1% triethanolamine in 0.5× PBTw.

• 19. In a 1.5-mL tube, combine 1.3 µL of acetic anhydride and 500 µL of 1% triethanolamine in 0.5× PBTw, and vortex briefly. Wash the embryos for 5 min in this solution.

• 20. Repeat Step 19.

• 21. Quickly wash the embryos twice with 500 µL of 0.5× PBTw.

Prehybridization

• 22. Repeat Step 21 one more time, but at the end of the wash, pass half of the 0.5× PBTw through the mini-column and replace it with room-temperature hyb buffer. Rock gently on a Boekel Rocker II for 20 min at room temperature.

  • The syringe remains attached during the washes in Steps 22–24. Ensure that the embryos remain submerged in liquid at all times by pulling back on the syringe plunger to form a vacuum in the mini-column.

• 23. Wash with 500 µL of room-temperature hyb buffer, rocking gently for 20 min at room temperature.

• 24. Wash with 500 µL of hyb buffer heated to 60°C in a water bath. Let the embryos stand in a hybridization oven for 2 h at 60°C.

Hybridization

• 25. Add DIG-labeled riboprobe to a final concentration of ∼0.5 µg/mL in 500 µL of hyb buffer. Incubate for 5 min at 100°C on a heat block.

• 26. Boil salmon sperm DNA, and shear by pulling in and out of a 25-gauge needle attached to a 1-mL syringe. Add 1.0 µL of 100 µg/mL boiled and sheared salmon sperm DNA to the DIG-labeled riboprobe solution.

• 27. Pass most of the buffer that the embryos are suspended in through the mini-column, and add the riboprobe solution.

• 28. Hybridize overnight at 60°C in a hybridization oven.

  • The syringe continues to remain attached during Steps 28–32. Ensure that the embryos remain submerged in liquid at all times by pulling back on the syringe plunger to form a vacuum in the mini-column.

Posthybridization Washes

• For posthybridization washes, all buffers should be preheated to 60°C and the embryos should be incubated in a 60°C hybridization oven.

• 29. Wash the embryos five times quickly and five times for 20 min each with 500 µL of plain hyb buffer.

• 30. Wash the embryos five times quickly and once for 30 min with 500 µL of 2× SSC plus 0.1% CHAPS/Tween.

• 31. Wash the embryos twice for 30 min each with 500 µL of 1× SSC plus 0.1% CHAPS/Tween.

• 32. Wash the embryos twice for 30 min each with 500 µL of 0.2× SSC plus 0.1% CHAPS/Tween.

Immunohistochemistry

• 33. Remove the embryos from the hybridization oven. Quickly wash the embryos twice with 500 µL of room-temperature 0.5× PBTw.

• 34. Incubate the embryos for 2 h at room temperature in 500 µL of blocking buffer.

• 35. Incubate the embryos overnight at 4°C in 500 µL of 1:1500 anti-DIG antibody:blocking buffer.

Post-Antibody Washes

• 36. Wash the embryos five times quickly and five times for 10 min each with 500 µL of MAB.

Detection

• Perform the transfers in Steps 39, 40, and 42 under a dissecting microscope.

• 37. Wash the embryos three times quickly and once for 10 min with 500 µL of AP developing solution.

• 38. Transfer embryos to a clean depression slide using a 9 inch glass Pasteur pipette.

• 39. Transfer all of the embryos to one well of a 96-well plate filled with 200 µL AP developing solution.

  • Transfer the embryos carefully; this may take some time. The embryos can remain in the well for up to 1 h.

• 40. Transfer the embryos to a well with 200 µL of Roche BM Purple AP substrate solution.

• 41. Develop in the dark at room temperature.

  • Development times vary; 4–16 h is typical for my probes.

• 42. To stop the reaction, transfer the embryos to a well filled with 0.5× PBTw.

  • The embryos can stay in 0.5× PBTw until they are mounted.

Imaging

• 43. Mount embryos on microscope slides in Fluoromount-G or other appropriate mounting medium.

• 44. Image on a compound microscope using DIC optics or on a laser scanning confocal microscope using specifications from Smith et al. (2016).

  • See Troubleshooting.