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*9.3.1 Labeling of embryo and transplantation of the embryonic shield

Keiji Inohaya~
Tokyo Institute of Technology

**9.3.1.1 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)

**9.3.1.2 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.

**9.3.1.3 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).

**9.3.1.4 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.

**9.3.1.5 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.

**9.3.1.6 References

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.
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