Embryonic Immunostaining 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× PBT.

  • 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.

• 4. Remove most of the liquid, leaving embryos suspended in ∼ 20 µL of 0.5× PBT.

• 5. 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.

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

Paraformaldehyde Fixation

• 7. 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.

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

Cleaning Mobicol Mini-Columns

• 9. 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× PBT 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.

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

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

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

• 13. 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

• 14. 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× PBT, followed by three quick washes with 500 µL 0.5× PBT.

Secondary Permeabilization

• 15. 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× PBT.

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

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

Primary Blocking

• 18. Wash the embryos three times for 10 min each and four times for 30 min each in 500 µL of 0.2% BSA.

• 19. Wash embryos twice for 30 min each with 500 µL of 5% NGS.

Primary Antibody

• 20. Add 500 µL of primary antibody diluted in 5% NGS, and leave overnight at 4°C.

  • The appropriate primary antibody concentration is antibody specific and needs to be experimentally determined. Typical antibody concentrations used for immunostaining are between 1:100 and 1:1500.

• 21. Wash three times for 5 min each and four times for 30 min each in 500 µL of 0.5× PBT.

Secondary Blocking

• 22. Wash two times for ½ h each in 500 µL of 5% NGS.

Secondary Antibody

• 23. Wash overnight with 500 µL of fluorescently labeled secondary antibody diluted in 5% NGS.

  • The appropriate secondary antibody concentration is antibody specific and needs to be experimentally determined. Typical antibody concentrations used for immunostaining are between 1:100 and 1:1500.

• 24. Wash three times for 5 min each and six times for 30 min each in 500 µL of 0.5× PBT.

Imaging

• 25. Mount embryos on microscope slides in Fluoromount-G or other appropriate mounting medium with anti-fade reagent.

• 26. Image with a fluorescence microscope.

  • See Troubleshooting.