Name: simple and effective administration and visualization of microparticles in the circulatory system of small fishes using kidney injection
Uploaded: 2018-06-17T15:00:00
Description: Watch this Scientific Journal Video about Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection at JoVE.com

著者は大幅ビデオ プロトコルの準備のために Bogdan Osadchiy とエヴゲニイ Protasov (ロシア、イルクーツク国立大学) の助けを認めます。この研究は、基礎研究 (#15-29-01003) ロシア科学財団 (#15-14-10008) とロシア財団によって支えられました。

すべての実験手順、動物実験のための EU 指令 2010年/63/EU に従い行った動物科目研究委員会の研究所の生物学イルクーツク州立大学によって承認されています。
1. マイクロ カプセルの作製
注: 蛍光染料を運ぶカプセルは帯電凝集沈殿処理7,8層によってアセンブリを使用して準備されます。すべてのプロシー?...

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ゼブラフィッシュ腎臓の微粒子による注入を示すためには、インジケーター染料搭載半透過性マイクロ カプセルを使用しました。したがって、プロトコルを含む逆荷電高分子電解質7,8,15,16,17 の層によってアセンブリを使用してマイクロ カプセルの作製について ,</su...

動物有機体にマイクロ サイズ粒子の管理は小さな光学センサー注入およびトランスジェニック細胞注入3、血管可視化2ワクチン配信1医薬品などの分野で重要な課題4,5します。 ただし、実験小動物の血管系へのマイクロ粒子の注入手順は難しい、特に繊細な水生生物です。ゼブラフィッシュのような人気のある研究標本のお勧めビデオ プロトコルを使用してこれらの手順を明らかにすること。
心内及びキャピラリーのマイクロインジェクション ゼブラフィッシュ血に microobjects の配信するためスタッフとユニークな手術設備が必要です。以前は、レトロな軌道手動注入3がセルを全体を管理するための簡単で効果的な方法として示唆されました。しかし、我々 の経験で目の毛細血管網の小さい区域のためかかりますこの手法から目的の結果を達成するために多くの練習。
本明細書で述べる循環系への堅牢で効率的な微粒子注入法大人ゼブラフィッシュの腎臓組織に直接手動注入による腎血管と毛細血管が豊富であります。この手法は、ゼブラフィッシュの腎臓6に細胞移植ビデオ プロトコルに基づいていますが、外傷性および時間のかかる顕微鏡手順が排除されました。提案手法は低死亡率によって特徴付けられる (0-20%) と高効率 (70-90% 成功)、それは一般に利用できる機器を使用して研究所に簡単。
提案プロトコルの重要な一部が注入された微粒子の (蛍光灯や色付きのもの) 場合ギル毛細血管でインジェクションの品質、数の大まかな相対評価の検証を可能にする可視化注入された粒子、循環血液から直接生理学的な測定のためスペクトル信号の検出。パク-1、最初に提案された Borvinskaya マイクロ カプセル化蛍光プローブを用いたゼブラフィッシュ血液 pH の生体内測定のためのプロトコルを示す記載した技術の可能なアプリケーションの例としてら。2017年5。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SNARF-1-dextran, 70000 MW</td>
			<td>Thermo Fisher Scientific</td>
			<td>D3304</td>
			<td>Fluorescent probe. Any other appropriate polymer-bound fluorescent dye can be used as a microcapsule filler</td>
		</tr>
		<tr>
			<td>Albumin-fluorescein isothiocyanate conjugate (FITC-BSA)</td>
			<td>SIGMA</td>
			<td>A9771</td>
			<td>Fluorescent probe</td>
		</tr>
		<tr>
			<td>Rhodamine B isothiocyanate-Dextran (RITC-dextran)</td>
			<td>SIGMA</td>
			<td>R9379</td>
			<td>Fluorescent probe</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>SIGMA</td>
			<td>C1016</td>
			<td>CaCO3 templates formation</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>SIGMA</td>
			<td>S7795</td>
			<td>CaCO3 templates formation</td>
		</tr>
		<tr>
			<td>Poly(allylamine hydrochloride), MW 50000 (PAH)</td>
			<td>SIGMA</td>
			<td>283215</td>
			<td>Cationic polymer</td>
		</tr>
		<tr>
			<td>Poly(sodium 4-styrenesulfonate), MW 70000 (PSS)</td>
			<td>SIGMA</td>
			<td>243051</td>
			<td>Anionic polymer</td>
		</tr>
		<tr>
			<td>Poly-L-lysine [20 kDa] grafted with polyethylene glycol [5 kDa], g = 3.0 to 4.5 (PLL-g-PEG)</td>
			<td>SuSoS</td>
			<td>PLL(20)-g[3.5]-PEG(5)</td>
			<td>Final polymer to increase the biocompatibility of microcapsules</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>SIGMA</td>
			<td>S8776</td>
			<td>To dissolve applied polymers</td>
		</tr>
		<tr>
			<td>Water Purification System</td>
			<td>Millipore</td>
			<td>SIMSV0000</td>
			<td>To prepare deionized water</td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>Stegler</td>
			<td></td>
			<td>For CaCO3 templates formation</td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Research&#160;plus pipette, 1000 &#181;L</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Dosing solutions</td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Research&#160;plus pipette, 10 &#181;L</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Dosing solutions</td>
		</tr>
		<tr>
			<td>Pipette tips, volume range 200 to 1000 &#181;L</td>
			<td>F.L. Medical</td>
			<td>28093</td>
			<td>Dosing solutions</td>
		</tr>
		<tr>
			<td>Pipette tips, volume range 0.1-10&#160;&#956;L</td>
			<td>Eppendorf</td>
			<td>Z640069</td>
			<td>Dosing solutions</td>
		</tr>
		<tr>
			<td>Mini-centrifuge Microspin 12, High-speed</td>
			<td>BioSan</td>
			<td></td>
			<td>For microcapsule centrifugation-washing procedure</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 2 mL</td>
			<td>Eppendorf</td>
			<td>Z666513</td>
			<td>Microcapsule synthesis and storage</td>
		</tr>
		<tr>
			<td>Shaker Intelli-mixer RM-1L</td>
			<td>ELMY Ltd.</td>
			<td></td>
			<td>To reduce microcapsule aggregation</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td></td>
			<td></td>
			<td>To reduce microcapsule aggregation</td>
		</tr>
		<tr>
			<td>Head phones&#160;</td>
			<td></td>
			<td></td>
			<td>To protect ears from ultrasound</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>SIGMA</td>
			<td>EDS</td>
			<td>To dissolve the CaCO3 templates</td>
		</tr>
		<tr>
			<td>Monosodium phosphate</td>
			<td>SIGMA</td>
			<td>S9638</td>
			<td>Preparation of pH buffers</td>
		</tr>
		<tr>
			<td>Disodium phosphate</td>
			<td>SIGMA</td>
			<td>S9390</td>
			<td>Preparation of pH buffers</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>SIGMA</td>
			<td>S8045</td>
			<td>To adjust the pH of the EDTA solution and buffers</td>
		</tr>
		<tr>
			<td>Thermostat chamber</td>
			<td></td>
			<td></td>
			<td>To dry microcapsules on glass slide</td>
		</tr>
		<tr>
			<td>Hemocytometer blood cell count chamber</td>
			<td></td>
			<td></td>
			<td>To investigate the size distribution and concentration of the prepared microcapsules</td>
		</tr>
		<tr>
			<td>Fluorescent microscope Mikmed 2</td>
			<td>LOMO</td>
			<td></td>
			<td>In vivo visualization of microcapsules in fish blood</td>
		</tr>
		<tr>
			<td>Set of fluorescent filters for SNARF-1 (should be chosen depending on the microscope model; example is provided)</td>
			<td>Chroma</td>
			<td>79010</td>
			<td>Visualization of microcapsules with fluorescent probes</td>
		</tr>
		<tr>
			<td>Fiber spectrometer QE Pro</td>
			<td>Ocean Optics</td>
			<td></td>
			<td>Calibration of microcapsules under microscope</td>
		</tr>
		<tr>
			<td>Optical fiber QP400-2-VIS NIR, 400 &#956;m, 2 m</td>
			<td>Ocean Optics</td>
			<td></td>
			<td>To connect spectrometer with microscope port</td>
		</tr>
		<tr>
			<td>Collimator F280SMA-A</td>
			<td>Thorlabs</td>
			<td></td>
			<td>To connect spectrometer with microscope port</td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>Fisherbrand</td>
			<td>12-550-A3</td>
			<td>Calibration of microcapsules under microscope</td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 22 mm</td>
			<td>Pearl</td>
			<td>MS-SLIDCV</td>
			<td>Calibration of microcapsules under microscope</td>
		</tr>
		<tr>
			<td>Glass microcapillaries Intra MARK, 10 &#181;L</td>
			<td>Blaubrand</td>
			<td>BR708709</td>
			<td>To collect fish blood</td>
		</tr>
		<tr>
			<td>Clove oil</td>
			<td>SIGMA</td>
			<td>C8392</td>
			<td>Fish anesthesia</td>
		</tr>
		<tr>
			<td>Lancet No 11</td>
			<td>Apexmed international B.V.</td>
			<td>P00588</td>
			<td>To cut the fish tail and release the steel needle from the tip of insulin autoinjector</td>
		</tr>
		<tr>
			<td>Heparin, 5000 U/mL</td>
			<td>Calbiochem</td>
			<td>L6510-BC</td>
			<td>For treating all surfaces that come in contact with fish blood during fish blood collection</td>
		</tr>
		<tr>
			<td>Seven 2 Go Pro pH-meter with a microelectrode</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>To determine fish blood pH</td>
		</tr>
		<tr>
			<td>Insulin pen needles Micro-Fine Plus, 0.25 x 5 mm</td>
			<td>Becton, Dickinson and Company</td>
			<td></td>
			<td>For injection procedure. Any thin needle (&#216; 0.33 mm or less) is appropriate</td>
		</tr>
		<tr>
			<td>Glass capillaries, 1 x 75 mm</td>
			<td>Hirschmann Laborger&#228;te GmbH &#38; Co</td>
			<td>9201075</td>
			<td>For injection procedure</td>
		</tr>
		<tr>
			<td>Gas torch</td>
			<td></td>
			<td></td>
			<td>To solder steel needle to glass capillary</td>
		</tr>
		<tr>
			<td>Microinjector IM-9B</td>
			<td>NARISHIGE</td>
			<td></td>
			<td>For precise dosing of microcapsules suspension</td>
		</tr>
		<tr>
			<td>Petri dishes, 60 mm x 15 mm, polystyrene</td>
			<td>SIGMA</td>
			<td>P5481</td>
			<td>For manipulations with fish under anesthesia</td>
		</tr>
		<tr>
			<td>Plastic spoon</td>
			<td></td>
			<td></td>
			<td>For manipulations with fish under anesthesia</td>
		</tr>
		<tr>
			<td>Damp sponge</td>
			<td></td>
			<td></td>
			<td>For manipulations with fish under anesthesia</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Thermo Scientific</td>
			<td>31212</td>
			<td>To remove the gill cover from the fish head</td>
		</tr>
		<tr>
			<td>Pasteur pipette, 3.5 mL</td>
			<td>BRAND</td>
			<td>Z331767</td>
			<td>To moisten fish gills</td>
		</tr>
	</tbody>
</table>

simple and effective administration and visualization of microparticles in the circulatory system of small fishes using kidney injection

生きている有機体にマイクロ サイズ粒子の全身投与は血管可視化薬とワクチンの配信、トランスジェニック細胞と小型の光学センサーの適用できます。しかし、生物学・獣医研究所の使用が多い、小動物に静脈内の薬剤が非常に困難な訓練された人員を必要とします。ここで、我々 は魚の腎臓への注入による微粒子のアダルト ゼブラフィッシュ (動脈分布) の循環系への導入のため堅牢で効率的なメソッドを示します。血管系で導入された微粒子を可視化、魚のえらに単純な生体イメージング手法を提案する.注入されたマイクロ カプセル化蛍光を使用して達成された体内監視ゼブラフィッシュ血液 pH のプローブ、パク-1、記載した技術の可能なアプリケーションの 1 つであります。この記事は、pH 感受性色素のカプセル化の詳細な説明し、迅速な注入の原理と体内蛍光信号の録音のための得られたマイクロ カプセルの可視化を示します。インジェクションの手法は低い死亡率率によって特徴付けられる (0-20%) と高効率 (70-90% 成功)、それは一般に利用できる機器を使用して研究所に簡単。グッピーやメダカなど、他の小さな魚の種にすべて記載されている手順を実行できます。

得られた結果は、提案するプロトコルの 3 つの主要なカテゴリのいずれかから来る: 蛍光色素 (図 1)、さらに可視化とマイクロ カプセルの腎臓射出カプセル化による蛍光微粒子の形成(図 2 および 3) 毛細血管を鰓と、最後に、生体内でスペクトルの記録を監視するパク-1 蛍光血液の pH のレベ...

Watch this Scientific Journal Video about Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection at JoVE.com

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

シンプルで効果的な管理と腎臓の注入を使用する小型魚の循環システムの微粒子の可視化

小型魚の circulatory system と魚血微粒子の体内可視化に蛍光微粒子の低侵襲、迅速注入の原理を説明します。

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Research

JoVE Journal

Biology

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To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

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蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

無傷の動脈の内皮層における一酸化窒素とスーパーオキシドのエン顔検出

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

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FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

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マウス単離された灌流腎臓テクニック

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

腎臓蛋白質の非ウイルス性発現の流体力学的腎盂注入

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

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Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...