この作業は、保健研究のカナダの協会、北のBCの血液研究所と大学のためのイノベーション、ブリティッシュコロンビア州センターの大学のためのカナダの財団によって運営されている。

1。試薬の調製<ol><li>実験全体で使用される試薬は、外科的プロトコルを開始する前に準備する必要があります。すべてのソリューションは、無菌操作を使用して行われたことに注意してください。 </li><li>基本的な塩溶液と炭酸水素ナトリウム溶液：生理食塩水を（PSS）ベストつのコンポーネントで用意されています。 4の20倍濃度と店舗℃で、両方のソリューションを準備する20Xでの重炭酸ナトリウムは360.0mMのNaHCO 3（84.01g/mol）であり、20X濃度で基本的な塩溶液は、2638.0mM塩化ナトリウム（58.44g/mol）、94.0mM塩化カリウム（74.56g/mol）、40.0mMのCaCl 2•2H 2ですO（147.02g/mol）、および23.4mMのMgSO 4•7H 2 O（246.48g/mol）。解決策は、前、次の化学物質を加えることに明確になるまでのソリューションは、一つずつ追加されるはずのdH 2 Oと化学物質で作製し、攪拌してください</li><li>重炭酸ナトリウムおよび基本的な塩溶液の等量を希釈し、1X濃度（のdH 2 O中の各重炭酸ナトリウムと塩基性塩のソリューションの2つのPSSの2L希釈100mLのために希釈することにより、PSSの2Lの準備</li><li> 37℃の水浴中にPSSとPSSを含む場所2Lのメスフラスコ。外科手術開始前1時間、5％CO 2 /バランスの窒素ガスでソリューションを平衡化する。 </li><li>血管作動性化学物質は、（例：フェニレフリン、アセチルコリン、ニトロプルシドナトリウム）最高-20℃でdH2Oで1M濃度に調製し、100μlのアリコートに格納されています</li></ol> 2。動物の準備<ol><li>マウスの重量を決定し、腹腔内注射によって90mg/kgでペントバルビタールナトリウムの初期投与量を管理する。手順ではマウスは、継続的につま先のピンチと呼吸数の精査を経由して麻酔の平面のために監視されます。麻酔の平面を維持するために20mg/kgで、必要に応じてペントバルビタールナトリウムの追加の注射を投与する必要があります。ノーザンBCの大学の動物実験委員会（ACUC）によって承認された動物のケアのプロトコルで説明されている麻酔の外科的平面は、連邦政府と制度の規定に従って維持されている。 </li><li>シェーバーで腹部と脇腹/ヒップエリアから毛を取り除く。アルコールに浸した綿棒で、残りの抜け毛を削除し、テープでエリアをパッティング。この実験的な手順は、端末であるため、これで十分です。 </li><li>仰臥位での明確な手術ボード上にマウスを置いて、ボードにそれぞれの足蹠をテープでボードにマウスを添付。体温は、外部の熱のランプを経由して維持され、その低体温は麻酔薬の結果として発生しないように直腸の温度計を介して監視されます。 </li></ol> 3。外科手術<ol><li>全体の外科手術中に曝露された組織が常に平衡加温したPSS溶液を用いて灌流保管されていることを確認します。手術器具との利益の血管に接触するか、またはそれらのまわりの脂肪組織や結合組織を操作することによって、それらに多くの力に置くことによって、血管を傷つけることにも重要です。 </li><li>解剖範囲の下にマウスを置きます。腹腔内の腹側表面に沿って正中切開を行い、マウスの脊柱に向かって皮膚を引っ込める。 </li><li>皮膚はLNが容易に見えるように収納されると、透明なSylgard 184の台座の上に皮膚をピン。 </li><li> LNの周りの領域を重ね合わせる結合組織の薄い層を削除します。 </li><li>脂肪組織のオーバーレイをオフにして、微小血管床が露出するまでリンパ節を囲む。メイン動脈セグメントはLNに隣接して産むと、一般的に静脈によってオーバーレイされる。全体の手術は約30分かかります。 </li></ol> 4。イメージングと血管の解析<ol><li>興味の血管セグメントは解剖範囲で露出されると、生体内顕微鏡で準備を確保する。動物はしばらくの間実験の期間中は、生体内顕微鏡で明確な手術ボード上に残っていることに注意してください。外科の項中に説明されているように体温は熱のランプを経由して維持され、直腸温度計によって測定される。セクション2.1で説明したように麻酔の面はまた、実験を通してモニターされる。 </li><li>灌流と吸引ラインを設定します。 PSSの灌流は、5％CO 2バランス窒素によって灌流されているウォータージャケット温水タンク（ウォータージャケットを循環水浴中で循環し、加熱される）、にと10ミリリットルの速度でドリップラインダウン、貯水池からの重力供給によって発生する/分。吸引ラインは、準備から灌流PSSを引く継続するために使用する必要があります。前の実験に吸引ラインとウォータージャケットが完全に洗浄され、各実験後に保存されていることに注意してください。これは、次の実験の前に無菌性を保証します。 </li><li> superfusing PSSの温度を確認してください。の温度を調整する° Cの準備を越え〜37の温度を達成するために水浴及び/またはPSS灌流の速度を循環。 </li><li>血管系は、少なくとも60分間PSSと平衡とビデオキャリパーを使用して血管の直径を定量化することができます。通常は、血管の直径は、容器が安定して確保するために（例えば、少なくとも40、50、および60分）複数のポイントで決定されるべきである。 </li><li>平滑筋特異的アゴニスト（例：フェニレフリン、PE）と内皮細胞特異的アゴニスト（例：アセチルコリン、アセチルコリン）を使用して、容器の健全性を評価する。アゴニストの濃度は、アゴニストに応じて調整する必要がありますが、PEとAchのための容器はsuperfusateに累積加算（10 - 9M〜10 - 5M）で各濃度で評価すべきである。各アゴニストのアプリケーションは、安静時の直径に船が戻るまでPSSを使用して洗浄の段階（通常は30分）で区切る必要があります。 </li><li>実験をトレース容器の健全性、その後の血管作用薬、蛍光標識、およびセルの次の評価を行うことができる。 </li><li>実験終了後動物をペントバルビタールナトリウムの過剰摂取を与えられます。動物は、つま先のピンチと呼吸運動や心臓の拍が不足しても応答がないことを保証するために監視されます。そのような時にこれを頸部脱臼が続いている。 </li></ol> 5。代表的な結果： 平衡期間は、次の準備には鼠径リンパ節を供給する細動脈と細静脈の両方の識別を可能にする明確なイメージを生成する必要があります。細動脈と明らかに血管の直径を計算するために重畳されるビデオのキャリパーのために観察される細静脈の壁を可能にするために船を囲む最小の脂肪細胞があるはずです。成功する準備の積分点では、細動脈の内腔を通って血管緊張のかなりの程度（すなわち、細動脈がvasoconstrictとそれぞれ筋肉と血管内皮特異的なアゴニストを滑らかにするvasodilateする能力を持っている）があることを移動する血​​液の豊富な供給を持っているです。 。 <img src="/files/ftp_upload/2551/2551fig1.jpg" alt="図1"/> 図1。生理食塩水と側面に沿って主要な静脈（緑の矢印）を実行しているで平衡化したリンパ節を供給鼠径リンパ節飼料細動脈（赤矢印）の生体顕微鏡像。 <img src="/files/ftp_upload/2551/2551fig2.jpg" alt="図2"/> 図2。濃度から生理食塩水に累積加算により灌流フェニレフリン（PE）への正常な血管作動性応答10 -9 M〜10 -5 M堅牢な血管収縮の存在は、動脈内に存在する通常の平滑筋の機能の示唆するものである。 <img src="/files/ftp_upload/2551/2551fig3.jpg" alt="図3"/> 図3。濃度10 -9 Mから10 -5 Mと生理食塩水に累積加算により灌流アセチルコリン（ACH）のノーマル血管反応堅牢な血管拡張の存在は、動脈内に存在する正常な内皮機能の示唆するものである。

<ol>
	<li>Bearden, S. E., Payne, G. W., Chisty, A., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteriolar+network+architecture+and+vasomotor+function+with+ageing+in+mouse+gluteus+maximus+muscle.">Arteriolar network architecture and vasomotor function with ageing in mouse gluteus maximus muscle.</a> J Physiol. 561, 535-545 (2004).</li><li>Looft-Wilson, R. C., Payne, G. W., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+expression+and+conducted+vasodilation+along+arteriolar+endothelium+in+mouse+skeletal+muscle.">Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle.</a> J Appl Physiol. 97, 1152-1158 (2004).</li><li>Payne, G. W., Madri, J. A., Sessa, W. C., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histamine+inhibits+conducted+vasodilation+through+endothelium-derived+NO+production+in+arterioles+of+mouse+skeletal+muscle.">Histamine inhibits conducted vasodilation through endothelium-derived NO production in arterioles of mouse skeletal muscle.</a> Faseb J. 18, 280-286 (2004).</li><li>Smeda, J. S., Payne, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+autoregulatory+and+myogenic+function+in+the+cerebrovasculature+of+Dahl+salt-sensitive+rats.">Alterations in autoregulatory and myogenic function in the cerebrovasculature of Dahl salt-sensitive rats.</a> Stroke. 34, 1484-1490 (2003).</li><li>de With, M. C., de Vries, A. M., Kroese, A. B., van der Heijden, E. P., Bleys, R. L., Segal, S. S., Kon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+anatomy+of+the+hamster+retractor+muscle+with+regard+to+its+microvascular+transfer.">Vascular anatomy of the hamster retractor muscle with regard to its microvascular transfer.</a> Eur Surg Res. 42, 97-105 (2009).</li><li>Domeier, T. L., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromechanical+and+pharmacomechanical+signalling+pathways+for+conducted+vasodilatation+along+endothelium+of+hamster+feed+arteries.">Electromechanical and pharmacomechanical signalling pathways for conducted vasodilatation along endothelium of hamster feed arteries.</a> J Physiol. 579, 175-186 (2007).</li></ol>

利害の衝突は宣言されません。

ここで紹介する鼠径リンパ節の生体顕微鏡は、生体内のリンパ節の画像の微小血管系に機能を提供しています。従って、それは直接、リアルタイムで観察する手段を容易にします。 LN微小血管系のイメージングは​​1つが免疫応答と血管の間のインターフェイスを研究することができるユニークなサイトです。この準備を使用して、フォーカスが免疫応答、血管系の変化で、または2つの間の相互作用で特に指示することができます。 すべての実験的アプローチと同様に、標準的な生体顕微鏡は、利点と限界の両方があります。ここで説明する準備のような標準IVMは、、簡単に修正することができる、と以前に蛍光トレーサーの染料または標識細胞集団の導入による落射蛍光顕微鏡法を可能にするために著者らによって実証されている。標準IVMは、3次元イメージングの可能性を与え、二光子顕微鏡または血管造影、細胞の追跡がまだであることが細胞間および細胞 - 血管系の相互作用を可能にする二次元で達成されることによって与えられることになるようトレースをしていませんが、観察し、定量化し、蛍光抗体での生体内投与し、その後の染色と組み合わせてリアルタイムでタンパク質/マーカーの発現に関するデータを提供するために使用することができます。 特に、代替のイメージング技術は、画像の静的な環境を必要とする、外科領域または平らな準備でカバースリップのほかのクランピングすることは頻繁に必要とされる。この制限は、、積極的に血管の整合性と生理学等またはIVMの準備中のように行わ血管拡張の実験のような他の手法の使用を評価するための準備を介して血管または他のメディエーターを灌流することを排除しない場合は。さらに、標準IVMは完全に平らな準備を必要とせず、そのような動物の呼吸によって生成されるように、運動の影響を受けません。これは、標準のIVMが高い再現性とより多くの外科領域で使用することができます。これは、ここで説明鼠径リンパ節の準備が例示される。リンパ節の大きさや形を考えると、準備が組織を傷つけることなく平坦にすることができないと、ノードの位置は、動物の呼吸のために重要な動きを生じさせる。このような問題は、他の方法で克服するのは困難ですが、簡単に説明した標準IVMの準備を使用して処理されています。 要約すると、上記の詳細製剤は、他の生化学、血管、および/またはそのような活性化または標識された細胞のサブセットの転送、低酸素症の誘導、および血管メディエーターの枯渇の過剰発現などの免疫学的手法は、任意の数と組み合わせることができます。しかし、追加のアプリケーションは、最初の準備の健康に依存しています。したがって、それは常にイメージングと評価されている血管の状態をテストすることが重要です。健康の準備を達成するためのキーポイントは、準備が常に体温で平衡化PSSで灌流して手術中に外科領域に配置接触し、ストレスを最小限に抑えている保証しています。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th></tr><tr><td>重炭酸ナトリウム</td><td>シグマアルドリッチ</td></tr><tr><td>塩化ナトリウム</td><td>シグマアルドリッチ</td></tr><tr><td>塩化カリウム</td><td>シグマアルドリッチ</td></tr><tr><td>塩化カルシウム二水和物</td><td>シグマアルドリッチ</td></tr><tr><td>ニトロプルシドナトリウム</td><td>シグマアルドリッチ</td></tr><tr><td>フェニレフリン</td><td>シグマアルドリッチ</td></tr><tr><td>アセチルコリン</td><td>シグマアルドリッチ</td></tr><tr><td>塩化マグネシウム七水和物</td><td>シグマアルドリッチ</td></tr><tr><td> Pentobarbitolナトリウム</td><td></td></tr></tbody></table>

intravital microscopy of the inguinal lymph node

体全体にあるリンパ節は、（LNの）、、免疫システムの不可欠なコンポーネントです。彼らはそれゆえ、適応免疫応答の誘導のためのサイトとして機能し、エフェクター細胞の開発。このように、LNSは、侵入病原体と戦うと健康を維持するための鍵となります。研究へのLNの選択は、アクセシビリティと希望するモデルによって決定され、鼠径リンパ節はよく位置しており、容易に皮膚および性器粘膜感染症の生物学的に関連するモデルの研究をサポートしています。 鼠径LNは、すべてのLNSのように、血でそれを提供する広範な微小血管のネットワークを持っています。一般的には、この微小血管ネットワークは高内皮細静脈（HEVのを）供給続いて枝とLNの主要な飼料細動脈が含まれています。ハイブリッド自動車は、恒常性と感染の両方の間にLNに免疫細胞の人身売買を容易にするために特化されます。ハイブリッド車は、このような状況の両方でLNに人身売買を規制する方法強烈な探求の領域です。 LNフィード細動脈、ハイブリッド車で直接上流の影響力を持っており、LNへの栄養素と細胞を豊富に含む血液の主な供給です。さらに、フィードの細動脈の変化は、適応免疫応答の誘導を促進に関与している。 LN微小血管系では、LNに最適な血液供給を維持し、適切なLN関数とその後の免疫応答において重要な要素であるLN、中に免疫細胞の流入を調節するのに明らかな重要性を持っています。 in vivoでの LN微小血管系を研究する能力は、免疫系および微小血管系の相互作用とLN内でお互いにどのように影響するかを解明する鍵となります。ここでは、鼠径リンパ節のin vivoイメージングのための手法を提案する。私たちは、健康な血管の研究、数時間にわたって実行可能な血管のイメージングを維持する能力、および容器の大きさの定量化を確保する方法に特に注意を払って、LNの微小血管のイメージングに焦点を当てる。血管作用薬だけでなく、携帯電話のトラフィックをトレースし、定量化する可能性のある微小血管の灌流のための方法も紹介されています。 鼠径LNの生体顕微鏡は、微小血管の機能と免疫細胞、LN、および微小循環の間に直接的なインタフェースのリアルタイムインタフェースを直接評価することができます。多くの免疫学的手法と同様に他のLNSの血管系を研究するために操作蛍光細胞標識と一緒に使われることが潜在的なこの手法。

Watch this Scientific Journal Video about Intravital Microscopy of the Inguinal Lymph Node at JoVE.com

Intravital Microscopy of the Inguinal Lymph Node

鼠径リンパ節（LN）の生体顕微鏡検査を行うための手法が説明されています。そのような技術は、リアルタイムを可能に<em&gt; in vivoで</em&gt;ホメオスタシスと感染時の両方のリンパ節の微小血管系と構造の研究。この手法は、細胞のトラフィッキングの研究にと他のリンパ節の部位に適応することができます。

鼠径リンパ節の生体顕微鏡

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

intravital-microscopy-of-the-inguinal-lymph-node

Research

JoVE Journal

Immunology and Infection

19.9K ビュー.  University of Northern British Columbia. 鼠径リンパ節（LN）の生体顕微鏡検査を行うための手法が説明されています。そのような技術は、リアルタイムを可能に in vivoでホメオスタシスと感染時の両方のリンパ節の微小血管系と構造の研究。この手法は、細胞のトラフィッキングの研究にと他のリンパ節の部位に適応することができます。

ビデオ: Intravital Microscopy of the Inguinal Lymph Node

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

広視野と共焦点顕微鏡によるマウスリンパ節スライスにおけるT細胞のex vivoでのイメージング

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

免疫・感染学

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

マウスの表在性リンパ節手術

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2光子顕微鏡を用いてマウスの胸腺の生体内イメージング

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

脾臓の生体顕微鏡検査：寄生虫モビリティと血流​​の定量的解析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

マウス膝窩リンパ節の生体内イメージング

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

胚中心の高解像度生体内ストライプ照明顕微鏡

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

ネズミリンパ節間質細胞の単離

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

以下の肝臓の生体顕微鏡イメージング<em&gt;リーシュマニア</em&gt;感染症：肝血行動態の評価

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

コレクションと RNA 解析のための大型動物からリンパ節の処理: リンパ節トランスクリプトーム研究大型動物種のための準備

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...