この作品は、(社) にドイツ研究振興協会 SFB688 によって支えられました。

マウス繁殖は Regierung Unterfranken によって承認された、すべてのドイツ語、欧州連合の法律、実験動物の管理と使用に関する規制に従い、すべての解析を行った。
1. コーティングのマイクロタイター プレート α4β1 インテグリンとEx 子宮尿膜培養
<ol><li>200 μ g/mL 滅菌リン酸緩衝生理食塩水 (PBS) で凍結乾燥させたマウス α4β1 インテグリンを再構成します。予め温めておいた (37 ° C) PBS で 10 μ G/ml の最終濃度に希釈します。ピペット希釈 α4β1 インテグリン溶液 20 μ L/ウェルのマイクロ プレート (1 尿膜/井戸) の井戸の必要な数に。 注: は、マイクロ プレートのウェルをコーティングする際、気泡の形成を避けるため。</li>
	<li>加湿の組織文化のインキュベーターでそれらを配置することによって 37 ° C で 60 分のマイクロタイター プレートを孵化させなさい。</li>
	<li>ピペットで上澄みを慎重に吸引します。穏やかな吸引を使用し、ピペット チップをウェル底面に触れることを避けるためにプレートを傾斜します。予め温めておいた (37 ° C) 培養液などダルベッコ変更イーグル培地 (DMEM)、50 μ L を追加することで 2 回洗浄し、穏やかな吸引によって洗浄媒体を削除します。</li>
	<li>重要なステップ: 無料サイトを 50 μ L/ウェル 0.1% (w/v) ウシ血清アルブミン (BSA) を追加することでバインド ブロック/DMEM 加湿組織文化のインキュベーターでマイクロタイター プレートを配置することによって ≥ 37 ° C で 30 分間インキュベートし、。 注: 尿膜外植体血清の存在下での様々 な面にアタッチできます。非特異的結合部位をブロックする必要したがって、コーティング後、インテグリンとの切り裂かれた尿膜を追加する前に井戸は、血清を含む文化を外植体中 (手順 4.4 を参照)。</li>
	<li>4.4 の手順で必要になるまでの 37 ° C でブロッキング液でコーティングされた井戸を維持します。</li>
</ol>2. 子宮解剖 Dpc 8 で妊娠マウスから
注: は、重量ゲイン差別15によって偽陽性妊娠率を減らします。
<ol><li>頚部転位によってマウスを犠牲に。腹部のコートを 70% (v/v) エタノールを噴霧して消毒します。 注: は、マウスを処理するとき、手袋を着用します。麻酔なしの頚部転位による安楽死は、尿膜添付ファイルを妨げる可能性があります尿膜の化学汚染を防ぎます。</li>
	<li>ハサミで正中線で小切開を行うことで腹部の皮膚を開きます。上と下の切開、皮膚を保持するために手袋をはめた指を使って、胸やマウスの尻尾に向かって皮膚を引き離します。</li>
	<li>鉗子で腹膜を把握します。もう片方の手を前方から V 字切開は後方に向かって、はさみを使用して、離れて腹腔内を公開する組織を移動します。子宮へのアクセスを得るために 1 つの側面に腸をプッシュするのに鉗子を使用します。</li>
	<li>2 つの子宮角を探します。鉗子で子宮頸部 (尾側のセグメント) を押しながら高級ハサミで靭帯をカットします。その後、はさみで卵管を切断することによって両方の子宮角をデタッチします。</li>
	<li>部屋の温度 (RT) PBS 滅菌、滅菌 10 cm 皿含むに子宮を転送する鉗子を使用します。脂肪組織、血管、神経周囲の高級ピンセット、はさみ (図 1 a) と子宮角を削除します。</li>
</ol>3. 胚解離
注: 8:1 ズーム範囲を備えた顕微鏡の下で次の手順に従います。
<ol><li>はさみで、着床間に切断することによって胚を区切ります。鉗子で子宮芽の筋肉子宮内膜をつかんでそれを分解、脱落膜 (図 1 b) を公開します。</li>
	<li>RT PBS 滅菌、含んでいる新しい 10 cm 皿に子宮芽を転送鉗子を使用して、します。</li>
	<li>鉗子の先端で胚の周り脱落膜を固定します。</li>
	<li>微細鉗子 (図 1)、脱落膜の抗 mesometrial 側に切開をして、胚 (図 1) を公開するための鉗子で、脱落膜をはがします。 注: mesometrial ポール脱落膜の高い血管柄付きし、胚が含まれますされ、淡い反 mesometrial 部分より根元が広くできます。</li>
	<li>鉗子 (図 1E, F) で胎児を解剖します。</li>
	<li>マイクロ スプーンを使用して、滅菌 3.5 cm 皿に含まれている滅菌 PBS のドロップに胚を転送します。</li>
	<li>マイクロ スプーンを使用して同じ 3.5 cm 皿の PBS の新鮮なドロップに胚を転送そして鉗子 (図 1, H) による卵黄嚢から胎児を解剖します。</li>
	<li>必要に応じて、必要な場合 (例えば、ポリメラーゼ連鎖反応による検出)、転送卵黄嚢生殖不能の管にそれを吸引によって ~ 大形オリフィス 200 μ L ピペット チップに 5 μ L の PBS。-20 ° C にてストア 注: 卵黄嚢準備を汚染する可能性が母体の組織 (すなわち、頭頂部の卵黄嚢ライヘルトの膜と胎盤のコーンなどを含む) は、誤ったジェノタイピングの結果につながります。これらの組織の除去は、卵黄嚢から胚解離の前に最も簡単です。</li>
</ol>4. 尿膜解離と固定化 α4β1 インテグリンのEx 子宮文化
<ol><li>微細鉗子 (図 1 H) による胚の後部の端にその基底挿入部位から尿をカットします。</li>
	<li>必要に応じて、発達のステージングが必要な場合は、段階の対照の光学と、5 ×、10 × 対物顕微鏡を用いた目視による胚の体節のペアをカウントします。 注: 胎児は体節を数えるための鉗子で拡張できます。漿の添付ファイルとの融合は ≥ 6 体節のペアを持つ胚で E 8.0 と 9.0 E の間発生します。6 8 体節のペアを持つ胚で回転を開始します。</li>
	<li>10% (v/v) ウシ胎児血清、L-グルタミン、プレコート マイクロタイター プレート井戸 (セクション 1 を参照) の必要な数の抗生物質と DMEM のピペット 40 μ L/ウェル。</li>
	<li>それを吸引によってフローティング尿を収集 〜 200 μ L ピペット チップの大形オリフィスに 5 μ L の PBS。1 尿膜/ウェルを転送します。</li>
	<li>例えば、加湿の組織文化のインキュベーター (37 ° C、5% CO2) でマイクロタイター プレートを配置することによって、allantoides をインキュベート 6、12、24 h。尿膜の添付ファイルとの干渉を防ぐためにこれらの時間ポイントの間にマイクロタイター プレートを移動しないでください。</li>
	<li>各時点でスコア尿膜添付ファイル (例えば、ない添付ファイル/フローティング尿膜部分的または完全にアタッチされた尿膜; 完全に広がり尿膜) 位相コントラストや微分干渉コントラスト光学系を搭載した顕微鏡を使用して、5 ×、10 × 目的 (図 2)。 注: 新鮮単離と完全に接続されている allantoides の代表的な例は図 2のとおりです。子宮 ex、野生型 c57bl/6 j マウスから分離した allantoides の遠位先端アタッチ (尿の部分的な添付ファイルとして呼ばれます) 4-6 時間、以内、全体の尿膜壁完全添付ファイルが可能です 〜 文化の 12 h。18-24 h 外植体が平坦化、CD31 陽性血管叢9,16(完全に広がった尿膜に呼ばれる) 円形の形状を想定します。</li>
</ol>5 血管内皮細胞マーカー CD31 Explanted 尿膜壁験染色
<ol><li>完全に広がり外植片の培養培地を吸引し、4% (w/v) パラホルムアルデヒド/PBS の 40 μ L/ウェルを追加してセルを修正します。 注: パラホルムアルデヒドは有毒です。手袋、皮膚と眼との接触を避けるために安全ゴーグルを着用します。</li>
	<li>常温では、10 分間インキュベートし、上清を吸引します。PBS の 40 μ L/ウェルを追加して 3 回を洗浄し、穏やかな吸引によって洗浄媒体を削除します。</li>
	<li>セルを permeabilize し PBS の 40 μ L/ウェルを追加することによって二度 0.1% (v/v) した洗浄で 30 分の PBS でトリトン X-100/0.1% (w/v) BSA/10% (v/v) ヤギ血清の 40 μ L/ウェルを追加することで非特異的結合部位をブロックし、穏やかな吸引によって洗浄媒体を削除します。 注: プロトコルを一時停止できる一晩この時点で。</li>
	<li>4 ° C で一晩に 20 μ L/ウェル主アンチ CD31 抗体の希釈 1:50 0.1% (w/v) BSA/PBS で追加することによって細胞を染色します。PBS の 40 μ L/ウェルを追加して 3 回を洗浄し、穏やかな吸引によって洗浄媒体を削除します。</li>
	<li>暗い室内で 0.1% (w/v) BSA/PBS アルミホイルで包装することによって配置の光から保護したマイクロタイター プレートで 1 時間で蛍光標識した二次ヤギ抗ラット抗体希釈 1: 200 の 20 μ L/ウェルを追加することでバインドされた一次抗体を検出します。PBS の 40 μ L/ウェルを追加して 3 回を洗浄し、穏やかな吸引によって洗浄媒体を削除します。</li>
	<li>メディアをマウントの 20 μ L/ウェルを追加することによって細胞を埋め込みます。インキュベート 〜 RT 取付培地が固化するまでで 1 時間。マイクロタイター プレート常温または 4 ° c、光から保護を格納します。</li>
	<li>5 ×、10 × 客観的かつ適切な蛍光フィルターを搭載した蛍光顕微鏡下で外植体画像尿膜壁血管叢 注: 完全普及尿膜の CD31 陽性血管叢の代表的なイメージは図 3のとおりです。Flk-1、Flt、に対して指示される抗体などの血管叢の他のマーカーとしてよく16ネクタイ 1 またはネクタイ 2 を使用できます。</li>
</ol>

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プロの接着細胞外マトリックス分子フィブロネクチンなどの豊富な源である血清の存在下で尿膜外植体は容易に様々 なプラスチックやガラス、フィルター表面9,16にアタッチされます。したがって、実行される分析の目的は、遺伝子組み換えの効果を具体的に検討する場合、尿膜の添付ファイルに他の処理固定化 α4β1 は、非特異的結合部位 (例?...

胎盤が子宮環境の胚の成長と開発のために不可欠です。それは内分泌と免疫器官としての酸素代謝物、胎児と母体の循環とも機能間の栄養交換のためのインターフェイスを構成します。胎盤発育を損なう内因性または外因性の侮辱は、子宮内胎児発育遅延、胎児の損失、または妊娠合併症、マウスと人間1の両方につながることができます。対象となるマウスを異常な胎盤の生理を引き起こす突然変異の数が 219 遺伝子多型のマウスのゲノム情報学 (MGI) ウェブサイトに現在表示されている2を増やす続けます (http://www.informatics.jax.org/vocab/ mp_ontology/MP:0010038)、これらの表現型の多くはよく機械論的視点から理解されていません。に加えて遺伝子変異、小分子薬、モノクローナル抗体、毒素、病原体、余分な代謝物や様々 な環境要因や影響を与える胎盤開発関数3,4,5,6,7します。 まだ、簡単な生体外モデル胎盤の開発で重要な手順が不足している試金。
(臍帯発達前駆体) 尿膜が絨毛膜8に向かって育つ芽として胚の後部の端から現れるとき、初期の経産婦でマウス胎盤の発育が開始されます。尿の芽の外側の層の Chorioadhesive セルは、添付ファイルおよび絨毛膜 (漿「融合」) の表面に尿膜の広がりを仲介します。絨毛膜はその後に尿膜壁血管成長迷路、栄養素と酸素が密接に並置母体血と胎児血管9 間交換される胎盤内層を形成する絨毛に折る ,10。
絨毛膜へ尿の付着迷宮形成の初期および重要なステップであり、このプロセスの欠陥は midgestation1胚性致死性の最も一般的な原因のうち。マウス突然変異体の数が、漿添付ファイル10が発生するが失敗し、MGI データベース現在異常な漿融合 (http:// によって特徴付けられる 108 マウスの突然変異体の一覧を記載されてwww.informatics.jax.org/vocab/mp_ontology/MP:0002824)、血管細胞接着分子 1 (VCAM 1、尿中胚葉に発現) と (は絨毛の中皮に表される α4β1 インテグリンの細胞間相互作用初期胚胚体外胚葉由来) 漿添付ファイルと融合11,12,13のため不可欠であることが表示されます。(E) 7.5 日約 VCAM 1 が尿の茎の遠位 3 分の 2 以内単位があることを示している全体マウント野生型胚の免疫組織化学的染色13 (1-4 体節期)、およびその発現状態のまま高漿の添付ファイルと - 融合11,12。絨毛膜尿愛着の障害は、通常、子宮の芽の組織学的解析による検出します。ただし、尿膜サイズはの間と同じ発達段階14リットル内でも高い生理学的変数であり、尿は、物理的な接触をするのに十分な大きさに達したときに絨毛膜にのみ添付できます。この自然の変動を反映して、漿添付が行われる間のいつか 〜 E 8.0 と 9.0 E子宮内、および組織によって、このプロセスの統計的信頼性の高い評価は大きい数の解析に依存したがって淘汰適切な発達段階で十分な検体を採取します。
ここでは、尿の添付ファイル子宮 ex少ない尿膜のサイズに依存している評価方法について述べる。妊娠マウスの子宮からマウス胚とその allantoides の郭清を紹介 〜 8 日のポストの coitum 定め(dpc; dpc 0.5 が膣にプラグインの検出を示す)、固定化 α4β1 の外植体尿膜の後続の文化とインテグリンです。このメソッドは、添付ファイルおよび複数並列で、異なる連続した時点で尿膜外植体の挙動を広がりの急速な機能評価できます。プロトコルを使用することがあります尿膜添付ファイル前のヴィヴォに及ぼすターゲット変異、薬、または様々 な環境要因の画面に。

<table border="0" cellpadding="0" cellspacing="0" width="1607">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">C57BL/6J wildtype mice</td>
			<td style="width:256px;">The Jackson Laboratory</td>
			<td style="width:119px;">Stock No:&#160;000664</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 % (v/v) Ethanol</td>
			<td style="width:256px;">VWR International</td>
			<td>930031006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>11000-12</td>
			<td style="width:915px;">Forceps used to open the abdominal cavity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Micro dissecting forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td>Dumont # 5 medical biology forcepts with fine, sharp tip.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fine scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>14094-11</td>
			<td>Straight blades.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>15012-12</td>
			<td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microspoon</td>
			<td style="width:256px;">Carl Roth</td>
			<td>AT18.1</td>
			<td>5 mm Spoon diameter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microtiter plates</td>
			<td style="width:256px;">ibidi</td>
			<td style="width:119px;">81501</td>
			<td>We use uncoated, sterile angiogenesis slides (internal volume 50 &#181;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-a4b1-mediated binding to plastic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">10 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">3.5 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150288</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large orifice pipette tips&#160;</td>
			<td style="width:256px;">Biozym</td>
			<td style="width:119px;">VT0140X</td>
			<td>Low binding pipette tips, 200 &#181;L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a4b1 integrin</td>
			<td style="width:256px;">R&#38;D Systems</td>
			<td style="width:119px;">6054-A4</td>
			<td>Murine a4b1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>Without Ca2+ and Mg2+.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:319px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-03600</td>
			<td style="width:915px;">The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO3 but no L-glutamine, which is added separately.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:319px;">Penicillin/Streptomycin</td>
			<td style="width:256px;">Gibco (ThermoFisher Scientific)</td>
			<td style="width:119px;">15140-122</td>
			<td>100 x (10,000 U/mL Penicillin and 10,000 &#181;g/mL streptomycin) stock solution.&#160;Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">L-Glutamine</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-80100</td>
			<td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fetal bovine serum&#160;</td>
			<td style="width:256px;">Biochrom</td>
			<td style="width:119px;">S 0115</td>
			<td>We use heat-inactivated FBS (heated to 56 &#176;C for 30 min with mixing to inactivate complement).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin (BSA)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">A7030</td>
			<td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &#181;m pore size and low protein-binding membrane, and store aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">para-Formaldehyde</td>
			<td style="width:256px;">Carl Roth</td>
			<td>0335.2</td>
			<td style="width:915px;">To make a formaldehyde solution in PBS, weigh para-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &#176;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &#176;C for approximately four weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Triton X-100</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">X100</td>
			<td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Normal goat serum</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>G9023</td>
			<td>To block non-specific binding of antibodies in immunofluorescence analyses.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a-CD31 Antibodies</td>
			<td style="width:256px;">BD Biosciences</td>
			<td>550274</td>
			<td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary goat anti-rat antibodies</td>
			<td style="width:256px;">Thermo Fisher</td>
			<td>A-11006</td>
			<td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Mowiol 4-88</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>81381</td>
			<td>Mounting medium for cytochemistry. MW ~31,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Labovert&#160;</td>
			<td style="width:256px;">Leitz</td>
			<td style="width:119px;">Labovert&#160;</td>
			<td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">M80</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">M80</td>
			<td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">DM4000B</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">DM4000B</td>
			<td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Digital camera</td>
			<td style="width:256px;">JVC</td>
			<td style="width:119px;">KY-F75U</td>
			<td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:319px;">Confocal microscope</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">SP5</td>
			<td style="width:915px;">Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td>
		</tr>
	</tbody>
</table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

胎盤は、成長と哺乳動物の胎児の開発のために不可欠です。このため、多数の遺伝子変異と胎盤の開発または機能を妨げる可能性が高いのも環境の侮辱が、マウスとヒトの妊娠初期の損失にあります。それにもかかわらず、画面の胎盤形成に対する潜在的な影響に簡単な生体外の試金が不足しています。ここでは、モデリングにおける胎盤絨毛膜に尿膜壁添付ファイルから成っている最初の重要なステップに焦点を当てます。Chorio 擬態の基板となる固定化 α4β1 インテグリンの尿外植片の添付ファイルを迅速に評価する方法について述べる。この体外アプローチにより、添付ファイルと異なる連続した時点で複数の尿膜外植体の挙動を拡散の定性的な評価です。プロトコルは、ターゲットを絞ったマウス突然変異、薬、または妊娠合併症や尿膜添付ファイル前のヴィヴォの胎児の損失にリンクされているさまざまな環境要因の効果を調べるために使えます。

このプロトコルを特定しマウス allantoides前のヴィヴォ、外植体について述べるし、体内漿融合中 α4β1 インテグリンに尿膜添付ファイル、重要なプロセスの定性的な評価の詳細。図 1 a-Hに示す代表的な結果は、妊娠子宮から始まって尿膜分離の連続した手順を示します。脂肪組織と血管 (図 1...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

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

モデル漿添付ファイル、胎盤形成の最初のステップの in vitroアッセイについて述べる。プロトコルは、固定化 α4β1 インテグリンのマウス allantoides の郭清と植の文化を示しています。尿膜の添付ファイルは、あらかじめ決められた時点で病理組織学的に評価されます。

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 ...

dissection-explant-culture-murine-allantois-for-vitro-analysis

Research

JoVE Journal

Medicine

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

7.8K ビュー.  University of Würzburg. モデル漿添付ファイル、胎盤形成の最初のステップの in vitroアッセイについて述べる。プロトコルは、固定化 α4β1 インテグリンのマウス allantoides の郭清と植の文化を示しています。尿膜の添付ファイルは、あらかじめ決められた時点で病理組織学的に評価されます。

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

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

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

プライマリーヒト線維芽細胞の導出のために皮膚パンチ生検植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

マウス大動脈洞におけるアテローム性動脈硬化症の病変の定量分析とキャラクタリゼーション

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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 ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

マウス前立腺マイクロ解剖と外科的去勢

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

の最適化分析<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

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 ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生児マウス蝸牛移植法<em&gt;インビトロ</em&gt;聴覚研究のスクリーニングツール

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

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 ...

郭清と尿の添付ファイルのIn Vitro解析マウス尿膜の培養

要約

さらに動画を探す

この動画の章

プロテオーム解析のための人間の硝子体の要素の解剖

プライマリーヒト線維芽細胞の導出のために皮膚パンチ生検植文化

マウス大動脈洞におけるアテローム性動脈硬化症の病変の定量分析とキャラクタリゼーション

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

Vitreodynamic分析のための全硝子体液解剖

マウス前立腺マイクロ解剖と外科的去勢

の最適化分析

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

新生児マウス蝸牛移植法

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

郭清と尿の添付ファイルのIn Vitro解析マウス尿膜の培養

要約

さらに動画を探す

この動画の章

プロテオーム解析のための人間の硝子体の要素の解剖

プライマリーヒト線維芽細胞の導出のために皮膚パンチ生検植文化

マウス大動脈洞におけるアテローム性動脈硬化症の病変の定量分析とキャラクタリゼーション

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

Vitreodynamic分析のための全硝子体液解剖

マウス前立腺マイクロ解剖と外科的去勢

の最適化分析

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

新生児マウス蝸牛移植法

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

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