MAK は、国立衛生研究所からこの作品のサポートを認めている/国立がん研究所許可番号 - 1R15CA152985 01A1。このプロジェクトは、マイアミ大学学部研究賞プログラム、奨学金プログラムとマイアミ大学夏学者プログラムにマイアミ大学博士課程工学部機会によってサポートされています。

ビデオで実施および下記の手順は、機関動物ケアおよび使用委員会 (IACUC) マイアミ大学によって承認されている。
 1。 準備と刺激テスト 手術、手術台、事後操作テーブルの <ol><li>確立する 2 つの明瞭な区域。すべての材料と必要な道具と両方のエリアをステージします。
	出されたボンネットの下 <ol><li>ステージ オペレーティング テーブル。プロシージャの継続的かつスムーズなパフォーマンスを可能にする方法で装備テーブルを配置します</li>。
		<li>は、同じ部屋および操作のメイン テーブルの近くで術後テーブルを確立します。プロシージャ全体で生殖不能の環境として、両方のテーブルを保持します</li>。
	</ol></li> <li>次の場所は、オペレーティング テーブルの上供給: 蓋、1 つ 15 mL チューブ、手術用はさみ、70% エタノール、2 つの発泡ボード、2 つの鉗子、シリンジ 1 mL 21 ゲージ、2 つの 50 mL チューブ、1 つ遠心分離機管の 1 つのスクイズ ボトルの 1 つのペアを 1 つのガラスの瓶、1 つのセラム、4 外科パッド、10 ピン、殺菌ワイプのディスペンサー、シャープス コンテナーします。</li>
	<li>は、後の操作テーブルの上次の消耗品を配置: 1 つ分析的なバランス、液体窒素、浅いワイド 4 L デュワー口デュワー、フローティング チューブ ラック、外科はさみのペアを 1 つ、2 つの鉗子、4 つのセラム チューブのディスペンサー 1滅菌ワイプします</li>。
	<li>ホルマリンで 1 つ 50 mL と 75% のボリュームに 1 つの 15 mL チューブを入力します</li>。
	<li>郭清ボードとしておおよそ 30 cm × 30 cm 発泡ボードに 1 つの外科パッドを貼る各コーナーで pin を使用します。操作中に残りの 4 つのピンを使用します。臓器を手術後テーブルに転送する外科パッドで小さい発泡ボードを準備します 。
	注意: イソフルラン (99.9%) は、有毒化学物質、排出される麻酔ガス 8 の清掃から安全性の最大レベルを確保するため口のフードで使用する必要があります。イソフルランを使用してオープン アンド ドロップ メソッドを使用してに関連付けられている研究者へのリスクに関する追加情報がテイラーとムック 8 記事</li>。
	<li>はガラスの瓶にイソフルラン (99.9%) を数滴に浸す外科パッドを配置し、マウス、イソフルランとの直接接触を防ぐために、上にペーパー タオルを配置します。同様に、残りの管ライン、イソフルランを数滴で吸収し、マウスと、イソフルランとの直接接触を防ぐために上に追加のパッドを配置する外科パッドを使用します</li>。
	<li>注ぐ液体窒素デュワー最大行を埋めるまでに達する浅い広口にします</li>。
	<li>マウスの麻酔に郭清のために選択の場所の部屋の すなわち 〜 1 分のふたで覆われてイソフルラン (99.9%) のいくつかを浸したパッド付きのガラス瓶が値下がりしました 
	注: 今回は、マウスから異なります。マウスが意識、商工会議所から削除し、操作盤の上に配置します</li>。
	<li>は、科学者から離れて指摘した頭と、腹側が横たわっているようにマウスを合わせます。イソフルラン (99.9%) の数滴を浸した外科パッドとライニング管の頭を置き、マウスの刺激に対して反応されるように足ピンチして刺激テストを実行します。
	<ol><li>場合は、このテストは失敗し、足ピンチ テストに対するマウスの応答、手順 1.8</li>。
	</ol></li></ol> 2。初期の切開、心臓穿刺、安楽死 <ol><li>手術の発泡ボードにマウスの四肢をピンし、70% のエタノールでマウスの腹側をウェットします</li>。
	<li>は、ピンセットで尿道口近くの毛皮をピンチし、若干上向きに引き出します。腹腔正中線を尿道口から始まって、あごで終了を手術用はさみで切開を行います。
	<ol><li>鉗子で毛皮の皮の 1 つの側面をつかむ最初の切開部の開始の点の近く、別の切開はさみで下方と斜めに背中足に向かってします</li>。
		<li>は、反対側に同じ方法でこれを繰り返します 。
		注: 毛皮の皮より広い開口部を作成する可能性がありますにピンを必要はありません</li>。
	</ol></li> <li>心を確認し、注射針の目詰まりを避けるために、心臓の周りの膜である心膜を取り外します。
	<ol><li>は、鉗子で心膜をつかんで、はさみでカットします。慎重に鼓動する心臓に注射針を挿入して心臓穿刺を行い、ゆっくりとピストンを撤回する開始します</li>。
		<li>最適な採血、心臓のポンプ作用を模倣し、あまりにもすばやく描画を避けるために針のプランジャーを使用します 。
		注: 通常約 1 mL の血液を収集することができます</li>。
		<li>血のコレクションを完了すると、遠心管とシャープス コンテナーにシリンジの処分に血を払拭します</li>。
		中心部を接続する添付ファイルを削除することによって安楽死実施 <li>心臓穿刺の実行後です 。
		注: ヘパリン、抗凝固剤は、この特定における血清コレクションの凝固する血を許可する心臓穿刺する前にこの手順で注射器に追加されませんでした。しかし、研究者は、プラズマを収集するために血液凝固を防ぐために場合は、ヘパリンに追加でした心臓穿刺前にシリンジします</li>。
	</ol></li> <li>研究には、マウスの遺伝子型が含まれている場合耳の部分をハサミでチョキンし、遺伝子型検証用遠心管に配置します</li></ol>。
 3. 膵臓領域抽出 <ol><li>マウスの左側にある胃を検索します。優しく (避けるために引き裂く) 開始 2 つの鉗子を使用して胃と十二指腸と膵臓を切り離すします 。
	注: 胃や腸から膵臓をデタッチする際、非常に重要な臓器から膵臓組織をガイドしていない粉砕したり、鉗子で膵臓を引き裂く優しく鉗子を使用は。
	<ol><li>小腸の空腸および回腸のセクションから膵臓を分離し続けると最後に大腸の盲腸から</li>。
	</ol></li> <li>、盲腸で鉗子を再配置および直腸に向けて残りコロンに沿って膵の分離を続行します 。
	注: この時点ではカットし、胃から結腸、直腸の直前の領域部分を削除すると便利です</li>。
	<li>膵臓と脾臓を添付します。マウスの右側に向かって膵臓をスライドさせます。隣接脾臓と膵臓を完全にデタッチする鉗子で膵臓と胸腔内の残りの関係を区切ります</li>。
	<li>は、膵臓を外し、検査のためにそれを広げ。識別目的のため膵臓に接続されている脾臓を残します。
	<ol><li>削除すべて結合組織、膵臓からの脂肪および腸間膜の組織 。
		メモ: この組織は色白く、こうして色でピンカーは、膵臓の組織から簡単に区別することができます。これは、膵臓を全削除する必要がある場合に特に重要です。たとえば、膵臓が圧迫され、体重と動物のグループ間比較する必要があります。Ptf1a cre/+;LSL Kras G12D/+ マウス モデル古いヶ月で特に硬線維性膵組織が存在する可能性があります。この場合、腫瘍組織で腸がインター レースにすると膵臓を慎重に除去を実施する必要があります。高度な場合は、異常な脾臓と肝臓組織もあるかもしれない</li>。
	</ol></li> <li>必要に応じて、この時点で他の臓器を削除します</li></ol>。
 4 データの収集とストレージ <ol><li>臓器の抽出後、サンプルを保全のため術後のエリアに移動します。</li>。
	<li>各器官の重量を量るし、彼らのそれぞれのセラムにそれらを配置します 。
	注: ウィットに沿ってh 質量今後の参考のため、各サンプルの任意の不正を記録すべきです</li>。
	<li>器官の重量を量ったが、一度スナップ凍結用液体窒素にそれらを配置します</li>。
	<li>凍結、スナップ後長期保存用-80 ° C で臓器を格納します</li>。
	<li>は一晩、ベンチ上ホルマリン保存サンプルを配置し、次の朝は 70% エタノールにホルマリンからのソリューションを変更します 。
	注意: これらのサンプルは、長期保存のための 4 ° C で保存する必要があります</li>。
	<li>長期保存、凍結-80 ° C で血と耳パンチ。血液から血清コレクション、30 分の凝固し、それを遠心血液を許可します。ピペットを使用して血清部分を削除し、-80 で格納 ° C</li>
</ol> 5。クリーンアップ 殺菌ワイプ <ol><li>サニタイズすべての解剖ツール。キャップ、イソフルランの管は手術パッドを浸した。新鮮な外科パッドと発泡ボードの外科パッドを交換してください。処分施設ごと採取されなかったマウスの部分 &#39; s 動物処分ポリシー</li>。
</ol>

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著者が明らかに何もありません。

既存のメソッドの意義 マウス解剖の他の非公式の動画が存在している間このビデオ説明が最初のプロの品質、査読、郭清10 でマウスの膵臓の収穫抽出に必要な詳細な手順のすべての視覚的なデモ.代謝とインスリンの主な器官をされている膵臓に生産、郭清と膵の収穫を生理的特性11の保存のためことができます。膵臓を分離することによっ?...

マウスはヒト膵癌1,2の重要な動物モデルとして登場しました。Ptf1acre/+;LSL KrasG12D/+マウス モデル、クリステンの膵臓、膵上皮内テロメラーゼ (PanINs)、その進歩と呼ばれる、膵臓の病変の開始に終って専らラット肉腫 (K ‐ Ras) 遺伝子が有効膵膵管癌は、一般的には PDACs3と呼ばれます。このマウス モデル システム、ヒト膵癌4、5生活や頻繁に PDAC に進捗状況の最初の 5 ヶ月以内、PanINs が出てくる追加の利点の最高の動物モデルの 1 つ、単一の年4、560-70 歳が人間の膵臓癌が最もよく起きるに対し。
Ptf1a から郭清による膵臓抽出cre/+;LSL KrasG12D/+パニン ・早期から進行 PDAC3,4まで様々 な年齢でのマウスは、膵臓癌の開発の詳細縦断的組織学的検討,5. 5 個から 15 ヶ月に至るまでの年齢層で膵臓の収穫も、準備組織健康から病気にかかったティッシュへの移行中に発生する膵4代謝の世界の変化の特徴を抽出する使用ことができます6,7。
この記事では、マウスの膵臓領域抽出の実行に必要な手順の完全な視覚的なガイドを提示し、膵臓がさらなる分析のための保存のためのガイドラインを提供します。個人を含む、その他の膵疾患の研究を行って I 型糖尿病、特に学生やマウスの膵臓を使っての収穫を含む研究に新しい捜査に役に立つはずのこのガイドは均等に貴重になりますメタボリック ・ プロファイリングまたは組織学的解析の郭清。

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Jar&#160;</td>
			<td style="width:288px;">Corning</td>
			<td style="width:119px;">3140-150</td>
			<td style="width:167px;">The glass jar used in the video has been discontinued.&#160; This is its replacement.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lid of Glass Jar</td>
			<td style="width:288px;">Corning</td>
			<td style="width:119px;">9985-150</td>
			<td>The glass jar lid used in the video has been discontinued.&#160; This is its replacement.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 mL Falcon Tubes</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:119px;">339650</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Surgical Scissors</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:119px;">9201</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Squeeze bottle&#160;</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:119px;">03-409-10DD</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">100% Ethanol</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:119px;">22-032-103</td>
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			<td height="21" style="height:21px;width:216px;">Formalin</td>
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			<td style="width:119px;">245-684</td>
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			<td height="21" style="height:21px;width:216px;">Foam Boards</td>
			<td style="width:288px;">Therapak</td>
			<td style="width:119px;">562908</td>
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			<td height="21" style="height:21px;width:216px;">1 mL 21G Syringes</td>
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			<td height="21" style="height:21px;width:216px;">50 mL Falcon Tubes</td>
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			<td style="width:119px;">339652</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2.0 mL Microcentrifuge Tubes&#160;</td>
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			<td style="width:119px;">02-681-258</td>
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			<td height="21" style="height:21px;width:216px;">Surgical Pads</td>
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			<td height="21" style="height:21px;width:216px;">T-Pins Length:&#160; 2&#34;&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Analytical Balance</td>
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			<td height="21" style="height:21px;width:216px;">4L Dewar</td>
			<td style="width:288px;">Taylor-Wharton</td>
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			<td height="21" style="height:21px;width:216px;">Shallow Wide Mouth Dewar</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:119px;">F3087-V</td>
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			<td height="21" style="height:21px;width:216px;">Floating Microtube Rack</td>
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			<td style="width:119px;">60986-100</td>
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			<td height="21" style="height:21px;width:216px;">Cryogenic Vial 1.2 mL, Sterile</td>
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			<td style="width:119px;">10-500-25</td>
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			<td height="21" style="height:21px;width:216px;">Isothesia (Isoflurane)&#160;</td>
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			<td style="width:119px;">050033</td>
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			<td height="21" style="height:21px;width:216px;">Liquid Nitrogen</td>
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			<td height="21" style="height:21px;width:216px;">Mouse Kras Strain</td>
			<td style="width:288px;">The Jackson Laboratory</td>
			<td style="width:119px;">OO8179</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse Cre Strain</td>
			<td style="width:288px;">MMRRC</td>
			<td style="width:119px;">OOO435-UNC</td>
			<td></td>
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dissection of the mouse pancreas for histological analysis and metabolic profiling

膵臓特定のトランスクリプション要因、1 a cre リコンビナーゼ; を調査しています。lox 停止-lox クリステン ラット肉腫、12 のコドンでアスパラギン酸グリシン (Ptf1acre/+;LSL-KrasG12D/+) ヒト膵癌のモデルとしてのマウスのひずみ。私たちの現在の研究の目的は、膵臓癌の進行の新規代謝マーカーを識別するためにです。尿、糞便、血液、膵臓組織の抽出物と癌の進行を舞台に膵臓の組織学的解析のメタボリック ・ プロファイリングを行った。マウスの膵臓人間のような明確に定義された固形臓器ではなく、むしろ簡単にマウス内部解剖学になじみのない個人またはほとんどまたはまったく経験を実施して個人識別はないびまん性分散軟部組織マウス臓器の解剖。この資料の目的は、詳細な測定を提供する郭清によりマウスの膵臓の除去には初心者を誘導する視覚的なデモ。この記事は特にメタボリック ・ プロファイリングまたは組織学的解析のため郭清によってマウスの膵臓の収穫が必要研究に学生や新しい研究者に貴重なする必要があります。

図 1動作環境領域の概要については、図 2のポスト操作領域を示しています。この設定は、機器の最小限の量を提供します、ステージング、個人はこの個々 のニーズに合わせて変更することができます。プロトコルは実験の特定のニーズに合わせて最適化する必要があります。この手順は、マウスの適切な euthanization<sup clas...

Watch this Scientific Journal Video about Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling at JoVE.com

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

このビデオの記事は、組織学的解析と代謝プロファイリングに対する郭清によってマウスの膵臓を正常に削除に必要な手順の詳細なデモを提供します。

マウスの膵臓の組織学的解析と代謝プロファイリングに対する郭清

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

dissection-mouse-pancreas-for-histological-analysis-metabolic

Research

JoVE Journal

Medicine

36.2K ビュー.  Miami University. このビデオの記事は、組織学的解析と代謝プロファイリングに対する郭清によってマウスの膵臓を正常に削除に必要な手順の詳細なデモを提供します。

ビデオ: Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

プロテオーム解析のための人間の硝子体の要素の解剖

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

医学

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

ヒト膵臓の収集プロトコル

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

Vitreodynamic分析のための全硝子体液解剖

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

内分泌と外分泌リプログラミングおよび増殖のためのモデルとしての膵管の連結を介して、マウスの膵臓への外科的損傷

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

組織学的分析のために筋肉凍結切片の調製でのやっていいことといけないことを行います

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

変更されたカフを用いた膵臓移植マウス モデル

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

マウスの心臓形態の心エコー検査・組織学的検討

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

郭清と尿の添付ファイルのIn Vitro解析マウス尿膜の培養

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortopathy β-アミノプロピオニ トリルによる大動脈瘤と解離のマウスモデルでのマイクロ CT 定量分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

免震を用いた消化管ホルモン分泌のメカニズム潅流ラット小腸

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

マウスの膵臓の組織学的解析と代謝プロファイリングに対する郭清

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