L. Maves, October 2003
I. Collect eggs.
Sort eggs into EM (p/s, f/s).
II. Prepare beads.
Beads should always be loaded fresh (the same day of the experiment). See specific protocols.
III. Dechorionation
Timing of this step will vary depending on when you need to implant. At about sphere stage, donor and host embryos are transferred into small 1.2% agar dishes in EM (p/s, f/s). Embryos are then manually dechorionated. At this time, some dishes can be moved to room temperature to slow growth, providing a wider window for having embryos at the proper stage for implantation.
III. Mounting
Place a drop of 2-3% methyl cellulose in Ringerıs (I use 2% for gastrula stages, 3% for post-gastrula) in the center of a transplantation-dedicated glass depression slide. Using a fire-polished pipette, transfer up to 10 embryos into the methyl cellulose and orient using a clean loop. Then flood the depression with Ringerıs (p/s, f/s). Pipette about 1ul volume of bead solution into the methyl cellulose.
IV. Implanting beads
I usually work on a UV stereomicroscope at about 100-150X, but have found that this procedure can be done on about any good dissecting scope. Using a pulled glass needle, sweep a bead through the methyl cellulose next to the area in the embryo where you want the bead. Use the needle to tear a hole in the skin. Then use the needle to push the bead so that it lies under some skin next to the cut-if it is not pushed inside and underneath skin, it is likely to get pushed out of the embryo when the skin heals. It is usually not necessary to remove any host tissue. Move on to the next embryo. After you have implanted a bead in all embryos on the slide, go back and check to see that all of the beads have remained in the embryos. If there is a significant number of beads left over on the slide, vacuum these up with a yellow-tip pipette (so that you donıt have a ³bath² of FGF, RA etc.)
V. Post-transplantation
Gently submerge the depression slide in EM (p/s, f/s) in a large petri dish. Avoid moving the slides or dishes for at least an hour or two.
Embryo Medium (p/s, f/s): to 1 liter of fresh Embryo Medium, add 20 ml of Sigma penicillin (5000 U)/streptomycin (5mg/ml). Filter-sterilize through a Nalgene 75mm filter unit.
Ringerıs (p/s, f/s): Following recipe in The Zebrafish Book:
23.2 ml 5M NaCl final 116 mM NaCl
2.9 ml 1M KCl final 2.9 mM KCl
1.8 ml 1M CaCl2 final 1.8 mM CaCl2
1.19 g Hepes final 5 mM Hepes
bring to about 1L with 2X gd water
pH to 7.2 with 1M NaOH
add 20 ml pen/strep and filter as for EM.
Glass needles: Sutter Instrument Borosilicate with Filament, OD 1.2mm, ID 0.69mm. Pulled using Program 69 on Westerfield puller (makes short, stiff needles).