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Part 1. Preparation of injection material
<ol>
 <li>Tg(globin:GFP) cells:</li>
 <li>Bleed adult donor fish using 10ul pipette tip coated in heparin (1unit/ul) to puncture fish behind gill. </li>
 <li>Aspirate red blood cells and dispense in cell buffer (0.9X PBS + 5% FBS + 1% Pen/Strep). </li>
 <li>Filter cell suspension over 40uM mesh and determine concentration of cells using hemocytometer as previously described by LeBlanc et al. </li>
 <li>Spin down and resuspend in cell buffer at desired concentration such that final injection volume is not more than 5ul. Here we inject 1.5-2 million cells/recipient. </li>
 <li>For the dextran, Texas Red&reg;, dissolve the dye in DPBS for a final injection concentration of 10-12 mg/mL. Plan to inject 4 uL per fish.</li>
</ol>
Part 2. Injection
<ol>
 <li>Anesthetize fish in tricaine (4.2ml (4mg/ml) tricaine/100ml fish water).</li>
 <li>Wash Hamilton syringe 3-4 times prior to injection using 70% ethanol. Rinse 3-4 times with 0.9X DPBS. </li>
 <li>Place fish dorsal side up and facing right on damp sponge.</li>
 <li>Hold the Hamilton syringe with your right hand and with your index finger on the plunger. Gently stabilize the body of the fish with your left hand.</li>
 <li>Position the needle with the bevel facing up such that if the fish s eye were a clock, the needle would be pointing at the 7:00 position and at a 45 degree angle to the fish (figure 1). </li>
 <li>Gently insert needle 1-2 mm into 7:00 position and slowly depress the plunger. </li>
 <li>Allow the fish to recover in fresh E3 water. </li>
 <li>Wash needle, as described above, between injections of different reagents.</li>
 <li>Flick cell solution to resuspend cells every few minutes to prevent cell clumping.</li>
 <li>Keep fish off flow for 1 week with daily water changes to avoid infection. We keep fish in ICU water for this period (10mL Stress coat + 5mL pemafix + 5mL melafix per 38 liters E3 water). </li>
</ol>
Part 3. Representative Results
When performed correctly, it is possible to visualize injected material if it has been labeled in some way. For example, Tg(globin:GFP) red blood cells should be seen under a fluorescent dissection microscope circulating in the vasculature of recipient casper fish soon after injection as shown in figure 2. Likewise, injection of a 70kDa dextran, conjugated to Texas Red&reg; can be visualized in the vasculature of transparent fish immediately following retro-orbital injection (figure 3). Fluorescent kidney cells from Tg(&beta;-actin:GFP) donor fish can also be injected retro-orbitally into irradiated casper recipient fish. These cells will eventually home to the recipient marrow (figure 4a) and may then go on to repopulate the kidney after several weeks (figure 4b).
If the injection is too shallow or the angle of the needle bypasses the retro-orbital venous sinus cavity, injected material may be visualized pooling around the eye or flowing out of it. Alternatively, if the injection is too deep, fish may experience tissue damage or excessive bleeding, though they may still recover. When performed correctly, mortality after retro-orbital injection should be less than 5% of total injected fish and success of delivery to the bloodstream should be greater than 90% of total injected fish.
<img src="/files/ftp_upload/1645/1645fig1.jpg" alt="figure 1" /> Figure 1: Illustration of retro-orbital injection technique. The right eye of the fish is represented as an analogue clock in which the seven o clock position corresponds to the correct injection spot. 
<img src="/files/ftp_upload/1645/1645fig2.jpg" alt="figure 2" /> Figure 2: Tg(globin:GFP) red blood cells circulating in the vasculature of the tail of an adult casper fish three days after retro-orbital injection. 
<img src="/files/ftp_upload/1645/1645fig3.jpg" alt="figure 3" /> Figure 3: Successful injection of a 70kDa dextran conjugated to Texas Red&reg; used as dye produces fluorescent vasculature in the transparent adult fish, casper and is readily viewed using standard whole animal fluorescence microscopy. 
<img src="/files/ftp_upload/1645/1645fig4a.jpg" alt="figure 4a" /> Figure 4a: Three days following retro-orbital injection of whole kidney marrow cells from Tg(&beta;-actin:GFP) donor fish, the cells can be visualized homing to the kidney marrow of irradiated adult casper recipient fish. 
<img src="/files/ftp_upload/1645/1645fig4b.jpg" alt="figure 4b" /> Figure 4b: Four weeks following retro-orbital injection of whole kidney marrow cells from Tg(&beta;-actin:GFP) donor fish, the repopulation of the recipient kidney with green donor whole kidney marrow cells can be visualized in irradiated adult casper recipient fish.

<ol>
	<li>Steel, C. D., Stephens, A. L., Hahto, S. M., Singletary, S. J., Ciavarra, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+lateral+tail+vein+and+the+retro-orbital+venous+sinus+as+route+of+intravenous+drug+delivery+in+a+transgenic+mouse+model.">Comparison of the lateral tail vein and the retro-orbital venous sinus as route of intravenous drug delivery in a transgenic mouse model.</a> Lab Animal. 37, 26-32 (2008).</li><li>Pinkerton, W., Webber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+injecting+small+laboratory+animals+by+the+ophthalmic+plexus+route.">A method of injecting small laboratory animals by the ophthalmic plexus route.</a> Proc. Soc. Exp. Bio. Med. 116, 959-961 (1964).</li><li>White, R. M., Sessa, A. S., Burke, C., Bowman, T., LeBlanc, J., Ceol, C., Bourque, C., Dovey, M., Goessling, W., Burns, C. E., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>LeBlanc, J., Bowman, T. V., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+whole+kidney+marrow+in+adult+zebrafish.">Transplantation of whole kidney marrow in adult zebrafish.</a> J. Vis. Exp. , (2007).</li></ol>

成人ゼブラフィッシュにおける眼窩注入は死亡率の最も低い発生率と血流に注入された材料の納入の最高効率が得られます。ために注射スポットの性質上、穿刺は感染症と失血の量の発生を減少させる、迅速に治癒に表示されます。サイトであっても繰り返して、例えば、薬物の毎日注射するための短い時間の間に注入されることがあります。 薬物の大量は、単純に浸?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Heparin sodium salt from porcine intestinal mucosa</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>2106</td>
<td>Preweighed vial of 300 USP units</td>
</tr>
<tr>
<td>Tricaine-S</td>
<td>&nbsp;</td>
<td>Western Chemical</td>
<td>&nbsp;</td>
<td>MS-222</td>
</tr>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Dextran, Texas Red&reg;</td>
<td>&nbsp;</td>
<td>Molecular Probes Inc.</td>
<td>D1830</td>
<td>&nbsp;</td>
<tr>
<td>E3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>5mM NaCl 
0.17 mM Kcl 
0.33 mM CaCl2 
0.33 mM MgSO2
</td>
</tr>
<tr>
<td>Microliter Syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>80300</td>
<td>701N 10uL SYR (26s/2&#x201D;/2)</td>
</tr>		</tbody>
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retro-orbital injection in adult zebrafish

動物全体の薬物治療は、薬理学的および化学的遺伝学的研究の任意のモデルシステムに不可欠なツールです。静脈内（IV）注射は、多くの場合、関心のエージェントの配信の最も効果的かつ非侵襲的なフォームです。ゼブラフィッシュの（<em&gt;ゼブラフィッシュ</em&gt;）、薬物の静脈内注入は長いので、小血管の直径の課題でした。これはまた、造血幹細胞移植時の細胞の注入のための重要なハードルを証明している。歴史的に、血流への注射は、心臓から直接行われた。心がしばしば感染、大量の失血または致命的な臓器の損傷を受けやすい魚を残して注入時の穿刺しかし、この内心臓手術は、非常に高い死亡率を持っています。マウスを使って私たちの経験を生かし、我々は、注射部位は、目の後ろと後眼窩静脈洞になっているゼブラフィッシュの新しい注入の手順を開発しました。この眼窩（RO）の注入法は、正常に成魚の薬の注入だけでなく、全体の腎臓の骨髄細胞の移植の両方で採用されている。 ROの注入は、従来の内心臓注射よりもはるかに低い死亡率を持っています。眼窩に注入されている魚は少なく、次の注入を出血する傾向があり、心臓などの主要な臓器に傷害の非常に低い危険にさらされている。さらに、適切に実行したとき、注入された細胞および/または薬物を迅速化合物は、そのニッチに簡単にホームに全体の魚と腎臓の細胞に対する効果を発揮できるように血流に入る。大人の魚の血流に材料の効率的な配信を可能にしながらこのように、この新しい注入法は、死亡率を最小限に抑えます。ここでは、Tgの眼窩注入することにより、この手法を例示する（<em&gt;グロビン</em&gt;：大人にGFP）細胞<em&gt;キャスパー</em&gt;魚だけでなく、赤色蛍光色素の注入（デキストラン、テキサスレッド<em&gt;</em&gt;）成人に<em&gt;キャスパー</em&gt;魚。私たちは、その後、全体の動物の蛍光顕微鏡で成功した注射を可視化する。

Watch this Scientific Journal Video about Retro-orbital Injection in Adult Zebrafish at JoVE.com

Retro-orbital Injection in Adult Zebrafish

ここでは、成人のゼブラフィッシュで眼窩の注入を行う方法を示しています。

アダルトゼブラフィッシュにおける眼窩インジェクション

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

retro-orbital-injection-in-adult-zebrafish

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JoVE Journal

Biology

24.5K ビュー.  Children’s Hospital Boston. ここでは、成人のゼブラフィッシュで眼窩の注入を行う方法を示しています。