Immunostaining protocol for marine zooplankton

  1. Fix larvae in 4% formaldehyde in PTW (PBS + 0.1% Tween-20) for 2 h.
  2. Dehydrate larvae through a graded series to 100% Methanol and store minimum over night at -20°C.
  3. After stepwise re-hydration to PTW, permeabilize with proteinase-K treatment (100 μg/ml in PTW, 1-3 min at room temperature, see Table in the in situ protocol).
  4. To stop proteinase-K activity, rinse larvae briefly (<1min) with Glycine buffer (5 μg/ml in PTW).
  5. Post-fix larvae in 4% paraformaldehyde in PTW for 20 min.
  6. Wash larvae 2 x 5 minutes in PTW.
  7. Wash larvae 2 x 5 minutes washes in THT (0.1 M Tris-HCl pH 8.5 + 0.1% Tween-20).
  8. Block larvae and antibodies (separately) in 5% sheep serum in THT (larvae for 1h at room temperature, primary antibodies for 1h at 4°C).
  9. Apply primary antibodies in THT with 5% sheep serum to larvae. Primary antibodies are used at a final concentration of 1 μg/ml for rabbit neuropeptide antibodies and at 0.5 μg/ml for mouse anti-acetylated tubulin antibody (Sigma, Saint Louis, USA).
  10. Incubate larvae over night at 4°C.
  11. Remove weakly bound primary antibodies by two x 10 minute washes in 1M NaCl in THT.
  12. Wash larvae 5 x 30 min in THT.
  13. Incubate larvae overnight at 4°C in the dark in 1 μg/ml anti-rabbit Alexa Fluor® 647 antibody (Invitrogen, Carlsbad, CA, USA) and in 0,5 μg/ml anti-mouse FITC antibody (Jackson Immuno Research, West Grove, PA, USA).
  14. Wash larvae 6 x 30 min with THT-buffer.
  15. Mount larvae in 87% glycerol containing 2.5 mg/ml of the anti-photobleaching reagent 1,4-diazabicyclo[2.2.2]octane (Sigma, St. Louis, MO, USA) (DABCO).

Note 1:
Treat larvae with shells, e.g. Pecten larvae, with 4% Paraformaldehyde in PBS with 50 μM EDTA pH 8,0 for 1h to decalcify their shells before the immunostaining procedure (performed as described above).
Note 2:
For stainings with Cnidarian larvae, use a mouse anti-tyrosylated tubulin antibody (Sigma, Saint Louis, USA) at 1 μg/ml, instead of a mouse anti-acetylated tubulin antibody.