Immunostaining protocol for marine zooplankton
- Fix larvae in 4% formaldehyde in PTW (PBS + 0.1% Tween-20)
for 2 h.
- Dehydrate larvae through a graded series to 100% Methanol
and store minimum over night at -20°C.
- 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
- To stop proteinase-K activity, rinse larvae briefly
(<1min) with Glycine buffer (5 μg/ml in PTW).
- Post-fix larvae in 4% paraformaldehyde in PTW for 20
- Wash larvae 2 x 5 minutes in PTW.
- Wash larvae 2 x 5 minutes washes in THT (0.1 M Tris-HCl pH
8.5 + 0.1% Tween-20).
- Block larvae and antibodies (separately) in 5% sheep serum
in THT (larvae for 1h at room temperature, primary
antibodies for 1h at 4°C).
- 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).
- Incubate larvae over night at 4°C.
- Remove weakly bound primary antibodies by two x 10 minute
washes in 1M NaCl in THT.
- Wash larvae 5 x 30 min in THT.
- 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).
- Wash larvae 6 x 30 min with THT-buffer.
- 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