9.3.1 Labeling of embryo and transplantation of the embryonic shield
Tokyo Institute of Technology
22.214.171.124 Equipment and reagents
- Glass tube (Model G-1, Narishige Co.)
- Pipette puller (PB-7, Narishige Co.)
- Grinder (EG-4, Narishige Co.)
- Microforge (MF-90, Narishige Co.)
- Biotin dextran (molecular weight 10,000; Molecular Probes): Biotin dextran is dissolved in 0.2M KCl to give 3% solution and the solution are centrifuged through a 20 µm pore filter.
- Partially purified hatching enzyme (Step1)
- 1.6% methylcellulose (4,000 cP) dissolved in the medaka Ringer
- ABC Vectastain Kit (Vector Laboratories, Inc.)
- ABC solution (avidin-biotinylated peroxidase complex in 1% DMSO, 0.1% Tween-20 in phosphate-buffered saline)
- AEC (3-Amino-9-ethylcarbazole; Vector Laboratories, Inc.)
- Avidin-fluorescein conjugate (NeutraLite, Molecular Probes)
126.96.36.199 Preparation of micropipettes
- Pull micropipette from glass tube with no capillary with a vertical microelectrode puller.
- Break the tips of the capillaries to the inner diameter 40 µm using a knife.
- Polish the tips of the capillaries with a grinder.
- Fashion a sharp spear-tip with a microforge.
188.8.131.52 Labeling of donor embryos
- Pull micropipette from capillary glass tubes with a vertical microelectrode puller.
- Break the tips of the capillaries slightly, and fill with the labeling solution (0.3% biotin dextran / 0.1% neutral red in 0.2M KCl)
- Inject the labeling solution into one of blastomeres at 1- or 4-cell stage by pressure. (The injected dye spread through intercellular cytoplasmic connections to all cells of blastoderm. Intravitelline injection of the dye does not label the medaka blastomeres.)
- Incubate the injected embryos until the early gastrula stage. (The embryos at an early blastula stage are kept at 4 ºC overnight. The exposure to 4 ºC had no adverse effect on development of medaka embryos).
184.108.40.206 Transplantation of the embryonic shield
- Dechorionate donor and host embryos with partially purified hatching enzyme before the early gastrula stage.
- Transfer the donor and host embryos on a depression slide in 1.6% methylcellulose.
- Under a stereomicroscope, draw cells of the embryonic shield directly into the pipette.
- Insert the transplanting pipette and transplant the labeled donor cells into the ventral margin of an unlabeled host embryo.
- Incubate the host and donor embryos in the medaka Ringer until appropriate stages.
220.127.116.11 Detection of the progeny of the transplanted cells
Whole-mount staining of labeled cells
- Fix the host embryos in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 ºC.
- Wash the specimens 3 times in PBS (10 min each).
- Incubated the specimens with ABC solution. avidin-biotinylated peroxidase complex in 1% DMSO, 0.1% Tween-20 in PBS for 2 hours at room temperature.
- Wash the specimens 3 times with 1%DMSO, 0.1% Tween-20 in PBS (15, 30 and 45 min each).
- Stain the donor-derived cells with AEC solution.
Detection of labeled cells in sections with fluorescence
- Fix the host embryos in 4% paraformaldehyde in PBS overnight at 4 ºC, and prepare the frozen sections.
- Incubate with the avidin-fluorescein conjugate in PBS containing 0.1% Tween-20 (PBST) at room temperature for 1 hour.
- Wash the sections in PBST
- Observe with a confocal microscope.
Inohaya K, Yasumasu S, Yasumasu I, Iuchi I and Yamagami K (1999). Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes. Develop. Growth. Differ. 41, 557-566
Westerfield M (1995). The Zebrafish Book (Ed.3). A guide for the laboratory use of zebrafish (Brachydanio rerio). Oregon: University of Oregon Press.
Kane DA and Kishimoto Y (2002). Cell labeling and transplantation techniques. In Zebrafish. Oxford University Press.