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(Source: S. O'Shea and M. Westerfield from Zebrafish Book 5th Edition)

Dechorionating & deyolking

1. Remove embryos from their chorions in batches of ~100 by placing in 1 mg/ml of pronase and swirling occasionally (5-10 minutes for 24 hpf embryos, 10-20 minutes for 3 day embryos). Finish dechorionation by gentle trituration using a Pasteur pipette. The chorions float and can be decanted. Rinse three times in cold Ringer's solution.

2. Transfer the embryos to cold Ringer's with EDTA and PMSF. Remove yolks by triturating with a glass pipette that has been drawn out to have a tip diameter approximately the size of the yolk.

3. Transfer the dechorionated, deyolked embryos to fresh, cold Ringer's solution and rinse twice.

4. Embryos can be frozen in liquid nitrogen and stored at 70°C at this point by transferring to a microcentrifuge tube and removing as much liquid as possible.

Solutions:

Pronase:

5 mg/ml pronase diluted to 1 mg/ml in Embryo Medium

PMSF:

Stock - 100 mM phenylmethylsulfonylfluoride in isopropanol. Immediately before use, add 30 μl of stock/10 ml Ringer's (final conc. 0.3 mM PMSF).

EDTA:

Stock - 10 mM EDTA, pH 7.0. Add 1 ml of stock/10 ml Ringer's (final conc. 1 mM EDTA)

Preparation of gel sample

1. Remove from freezer and thaw the frozen, dechorionated, deyolked fish.

2. Microfuge for 1-2 minutes to pellet.

3. Remove excess liquid.

4. Add 150-200 μl SDS sample buffer (for example, ~50-100 3 day embryos or 100-150 24 hpf embryos; this will yield enough for a 1.5 cm curtain or four 0.4 cm lanes of about 25-30 μl each).

5. Homogenize with microfuge pestle until uniform in consistency.

6. Repeat step 5 until sample is no longer stringy.

7. Boil 5 minutes in a water bath.

8. Microfuge, 1-2 minutes.

9. Transfer supernatant to a new microfuge tube. Discard pellet or add more sample buffer and homogenize again if significant pellet remains.

10. Freeze at -70°C or run immediately on gel.

Solutions:

SDS sample buffer:

0.63 ml 1M Tris-HCl, pH 6.8
1.0 ml glycerol
0.5 ml β-mercaptoethanol
1.75 ml 20% SDS
6.12 ml H2O
(10 ml total)

Store at -20°C in aliquots.

For cytoskeleton/extracellular matrix preps:

Add an extraction step between steps 3 and 4 (above).

1. Homogenize dechorionated, deyolked embryos in extraction buffer.

2. Incubate overnight at 4°C.

3. Centrifuge 20 minutes at 5000 x g.

4. Remove supernatant.

Solutions:

Protein extraction buffer:

10 mM Tris, pH 7.4
2% Triton X 100
1 mM PMSF
1 mM aprotinin
1 mM leupeptin
1 mM trypsin inhibitor

5. Load, run and transfer gel

Immunoblotting (using PVDF blotting paper)

1. After the antigen is blotted, immerse the membrane at a 45° angle, into the blocking solution. Gently agitate for 60 minutes at room temperature or overnight at 4°C.
2. Decant the blocking solution and add TTBS to the membrane. Wash for 10 minutes with gentle agitation at room temperature.

3. Decant the TTBS and add the primary antibody diluted in 1% dried milk in TTBS. Incubate 4 hours at room temperature (or overnight at 4°C) with gentle agitation.

4. Remove the unbound 1° antibody by washing twice for 5 minutes each in TTBS.

5. Add alkaline-phosphatase conjugated 2° antibody solution (diluted 1:3000 in 1% dried milk in TTBS). Incubate 1-2 hours at room temperature with gentle agitation.

6. Wash membrane twice for 5 minutes each in TTBS and then, just before color development, once for 5 minutes in TBS to remove the Tween-20.

7. Immerse membrane in color development solution. Proteins present at 100 ng or greater will immediately become visible as purple bands. Lower amounts will take longer, but should be visible within 30 minutes. Staining can continue up to 4 hours.

8. Rinse membrane four times for 5 minutes each in dH2O.

Solutions:

Blocking solution:

3% dried milk in TBS

TBS:

20 mM Tris, pH 7.5
500 mM NaCl

TTBS:

20 mM Tris, pH 7.5
500 mM NaCl
0.05% Tween-20

Western Blot Color Development Solution:

66 μl nitroblue tetrazolium (NBT) stock
33 μl 5-bromo-4-chloro-3-indolyl galactopyranoside (BCIP) stock
10 ml color development buffer
Both NBT and BCIP stocks are 50 mg/ml in dimethylformamide. NBT stock is made by suspending 50 mg NBT in 700 ml dimethylformamide. Vortex. Add 300 μl distilled water to dissolve. Store both stocks at 4°C in dark.

Western Blot Color Development Buffer:

102 mg MgCl2
4.2 g NaHCO3
500 ml dΗ2O

7 Comments

  1. Anonymous

    Which ringer's solution should we use for de-yolking the embryos? Normal, high calcium or calcium free?

    Thanks in advance,

    Suzanne, PhD at Leiden Medical Center.
    Mail: s.j.duivesteijn_REPLACETHISWITHANAT_LUMC.nl

    1. I recommend the calcium-free ringers solution.  Low Ca2+ softens up the tissue, making it easier for the yolks to come off with gentle trituration.  By using calcium-free ringers with EDTA (as in the protocol), you will reduce free Ca2+ to very low levels.

      Dave Fashena
      ZFIN curation group

  2. Anonymous

    hello,

    at what step protease and phosphatase inhibitor should be added?

    Ludo, PhD  @ the IMB Brisbane

    1. The serine-protease inhibitor PMSF is added in step 2.  In this protocol, the other protease inhibitors are used only in an optional protein extraction procedure recommended for cytoskeleton/extracellular matrix preps.  This optional procedure is performed before Step 4 (addition of SDS sample buffer).  

  3. Anonymous

    Anonymous 

    Hi, I am a high school student in the 10th grade trying to do a project on cryopreservation of zebra fish embryos using liquid nitrogen then reviving it. Would this be the right thing for me to do. If not can you please help me to find the right information for me to get on the right path.

    e-mail address: snowberys@yahoo.com 

    1. This is a very interesting idea for a project.  In general, the challenge is always to find some tractable sub-project where you can pose a well-defined hypothesis and then test your hypothesis in a rigorous way.  

      As you may already have learned, it's a lot easier to cryopreserve germ cells than whole embryos.  Cryopreserving embryos is a tough (and very interesting) scientific problem and a lot more work needs to be done before science can catch up with science fiction.  

      For background, you might start with a publication search for "cryopreservation zebrafish" at NCBI PubMedhttp://www.ncbi.nlm.nih.gov/pubmed?term=cryopreservation%20zebrafish

      additional background - some websites with related work by Dr. Mary Hagedorn on cryopreservation:

      http://nationalzoo.si.edu/aboutus/staff/biosandprofiles/hagedornmary.cfm

      http://nationalzoo.si.edu/aboutus/staff/biosandprofiles/hagedornmary.cfmhttp://www.smithsonianmag.com/science-nature/Saving-CoralThrough-Sperm-Banks.html

      http://nationalzoo.si.edu/scbi/ReproductiveScience/FishCryoPres/default.cfm

      You might also try posting a query to the Zebrafish Newsgroup Information, instructions below:

      http://zfin.org/zf_info/news/newsgroup.html

      Good luck!

      Dave Fashena, ZFIN curation group

      p.s. for future reference, this question would be more appropriately placed in one of the cryo- sections of the Genetic Methods part of the protocols wikihttps://wiki.zfin.org/display/prot/Genetic+Methods