Pygmy Squid Whole-Mount In Situ Hybridization

This protocol is largely based on Yu and Holland (2009) with some modification. Some procedures followed Ono et al. (2014) and Yoshida et al. (2010). For further information, please refer to the papers. The embryonic stages of pygmy squid embryos were defined according to Yamamoto (1988). 

Pygmy squid Whole-Mount In Situ Hybridization (Ver 1.0 by Hiroki Ono)

Fixation and Dehydration

Fixation

1. Peel off fertilization envelope with using sharpened forceps. Especially for stage 21 and younger, pre-fix with Bouin’s fixative (< 1min) will make peeling off easier.

2. Transfer the embryos to a new 1.5ml microtube. Remove sea water (prevent from squash embryos, it is not necessarily to remove all liquid) and add 500 µl of PFA-ME.

3. Remove liquid and add 1ml of fresh PFA-ME

4. Fix the embryos for overnight at 4˚C. (For stage 24 and older, 2 days of fixation may work well.)

Dehydration (All dehydration steps should be proceeded on ice.)

5. Remove the PFA-ME, and wash the embryos with 500 µl of ice-cold 100% ethanol for 5min.

6. Wash the embryos twice with 1ml of ice-cold 100% ethanol of methanol twice for 30min each. Store them at -20˚C in a freezer (a non-self-defrosting freezer would be better).

Pretreatment (Re-hydration to pre-hybridization)

1. Add 400 µl of 100% ethanol or methanol to well of multi-well culture plate. Transfer fixed embryos to each well.

H2O2 treatment (optional, for stage 24 embryos and older)

2. Remove liquid and, and immediately (prevent from drying up) add 6% H2O2/ethanol or methanol (100 µl of 30% H2O2 + 400 µl of 100% ethanol or methanol). Let sit for 30 min. (At this point, most of pigments in the body still remains but they would fade away during following procedures. However, it is really hard to bleach the pigmentation in eyes completely.)

3. Add 100 µl of PBST-EDTA and mix gently for 5 min (At this point, the concentration of ethanol would be 80%.)

4. Add 300 µl of PBST-EDTA and mix gently for 5 min (At this point, the concentration of ethanol would be 50%.)

5. Add 800 µl of PBST-EDTA and mix gently for 5 min (At this point, the concentration of ethanol would be 25%.)

6. Remove as much liquid as possible and wash the samples gently twice for 5 min each with 1ml of PBST-EDTA.

HCl treatment (Optional)

7. Treat the samples with 0.2N HCl /PBST-EDTA (200 µl of 1N HCl + 800 µl of PBST-EDTA) for 5-10 min. (For stage 18 embryos and younger, HCl treatment is not needed; for stage 20-23 embryos, treat for 5 min; for 24-27 embryos, treat for 8 min; for 28 embryos and older, treat for 10 min.) After treatment, wash the embryos three times for 5 min with PBST-EDTA.

Permeabilization (Choose 9 or 10)

8. Permeabilize the samples by treating with RIPA Buffer (Wako, Code No. 182-02451) for 30 min.

9. Permeabilize the samples by digesting with 500 µl of 10µg/ml proteinase K/PBST-EDTA. (For stage 18 embryos and younger, digest for 5 min; for stage 20-23 embryos, digest for 8 min, for stage 24-27 embryos, digest for 10 min; for stage 28 embryos and older, digest for 15 min.)

10. Wash the samples twice for 5 min each with 500 µl of PBST-EDTA.

11. Re-fix the samples with 500 µl of 4%PFA/PBST-EDTA for 30 min.

12. Wash the samples twice for 5 min each with 500 µl of PBST-EDTA.

Pre-hybridization

13. Rinse the samples in 500 µl of hybridization buffer for 3 min. Transfer the samples to a fresh 500 µl of hybridization buffer, and cover the plate, and prehybridize at 60˚C in a hybridization oven with gentle rocking for 2 h.

Hybridization

1. Prepare hybridization buffer containing appropriate concentration* of RNA probe. (*The concentration is dependent on the gene expression level, the length of RNA probe and the quality of RNA probe. Normally, for DIG-labeled RNA probe, 100-200ng/ml in hybridization buffer will work well.) Denature the probe(s) by heating in a heat block for 5min at 70˚C.

2. Remove as much liquid as possible and add 500 µl of pre-heated hybridization buffer with the probe.

3. Hybridize in a hybridization oven at 60˚C with gentle rocking for more than 16 h.

Washes and Incubation with Anti-DIG-AP Solution

1. Transfer hybridization buffer with RNA probe to a new 1.5ml microtube. (This hybridization buffer can be reused several times.) Add 500 µl of pre-heated fresh hybridization buffer without RNA probe and incubate in a hybridization oven for10 min at 60˚C with gentle rocking.

2. Wash three times for 20 min each with 500 µl of pre-heated washing solution in a hybridization oven at 60˚C with gentle rocking.

3. Wash twice for 10 min each with 500 µl of pre-heated MABT in a hybridization oven at 60˚C with gentle rocking.

4. Wash twice for 5 min each with 500 µl of MABT at room temperature.

5. Remove as much liquid as possible. Add 500 µl of 2% blocking reagent and mix gently for 1 – 2 h at room temperature. (A horizontal orbital shaker will work well for mixing. Otherwise, mix gently every 10 min.)

6. Wash twice for 3 min each with 500 µl of MABT at room temperature.

7. Remove as much liquid as possible and add 500 µl of anti-DIG-AP solution, and incubate overnight at 4˚C (On a horizontal orbital shaker will work for a better staining without unevenness).

Washes and Staining

1. Transfer anti-DIG-AP solution to a new 1.5 ml microtube. (The anti-DIG-AP solution can be stored at -20˚C and reused several times.)

2. Wash the samples 6 times for 30 min each with 1 ml of MABT at room temperature on a horizontal orbital shaker (or mix gently every 10 min).

3. Wash the samples 3 times for 5 min each with 500 µl of AP buffer at room temperature on a horizontal orbital shaker.

4. Remove as much liquid as possible and add 500 µl staining solution (AP buffer plus appropriate amount of BCIP / NBT solution). Place the samples in the dark. Check the color development sometimes under a dissecting microscope.

Stop staining and store samples

5. When the staining develops to enough intensity, remove as much liquid as possible and rinse the samples with 500 µl of MABT for 5min.

6. Post-fix with 500 µl of 4%PFA/PBST

7. Rinse the samples twice for 3 min each with 500 µl of PBST.

8. Transfer the samples to the 60% glycerol/PBST solution.

Buffers

5x MOPS-EGTA(ME)

0.5M MOPS, 10mM MgSO4, 5mM EGTA, 2.5M NaCl

PFA-ME

4% Paraformaldehyde, 1x MOPS-EGTA

PBST-EDTA

1x PBS, 0.1% Tween 20 (or 0.1% Triton-X 100), 5mM EDTA

Hybridization buffer

50% formamide, 100µg/ml heparin, 100µg/ml Yeast RNA, 5x SSC, 5mM EDTA, 0.1% Tween 20.

5x MAB

500mM Maleic acid, 750mM NaCl, adjust the pH to 7.5 with NaOH pellets.

MABT

1x MAB, 0.1% Tween 20

2% blocking reagent (10ml)

0.2g blocking reagent (Roche), 2ml 5x MAB (the final concentration will be 1x), add H2O to 10ml.

Anti-DIG-AP solution

Add 1µl of Anti-DIG-AP (Roche) to 3ml of 2% blocking reagent.

References

1. Yu JK, Holland LZ. Amphioxus Whole-Mount In Situ Hybridization. Cold Spring Harb Protoc. 2009 Sep;2009(9):pdb.prot5286. 

2. Ono H, Kozmik Z, Yu JK, Wada H. A novel N-terminal motif is responsible for the evolution of neural crest-specific gene-regulatory activity in vertebrate FoxD3. Dev Biol. 2014 Jan 15;385(2):396-404.

3. Yoshida MA, Shigeno S, Tsuneki K, Furuya H. Squid vascular endothelial growth factor receptor: a shared molecular signature in the convergent evolution of closed circulatory systems. Evol Dev. 2010 Jan-Feb;12(1):25-33.

4. Yamamoto M. Normal Embryonic Stages of the Pygmy Cuttlefish, Idiosepius pygmaeus paredoxus Ortmann. Zool Sci. 1988 5:989-998