我々は、試薬の準備の支援のためにレベッカのPaquetteを感謝します。 MPMは、国立衛生研究所からの補助金からの健康と支援の国立研究所GM - 41514（MDC）、NS - 48252（KGC）、GM - 48071（IP）からルースL. Kirchstein博士号を取得する前のフェローシップでサポートされています。

1。細胞の養子移入： 尾静脈または眼内注射は、細胞の導入トラッカー標識または蛍光タンパク質を発現するリンパ球の両方に適した方法である。このビデオで。我々は、尾静脈注射によって細胞の養子移入を示しています。 A.材料 CD4 + T細胞を37℃で30分間、1.4μMのCFSEで標識されていますが、2回洗浄し、RPMI - 1640の100μLに再懸濁した。細胞は、無菌の注射用28ゲージのインスリン注射器にロードされます。使用する細胞の数は、実験が行われているに依存します。通常は、500万T細胞は、単純なT細胞の可視化のために十分です。マウスのための齧歯類の現像抑制剤は、動物に、自分自身への負傷を​​防止するために必要とされる。 B.動物の保護マウスをゆっくり（ビデオを参照）げっ歯類の制止する人に動物をバックアップし、静かにプランジャーを押すことにより管の開放端を閉じることによって養子移入のために確保される。いくつかの小さな動物が制止する人の内部に好転することができるように、このプロセスの間にマウスの尾から行くようにしてください。このメソッドは、自分自身を傷つけるからと注入中に移動するから動物を防ぐことができます。げっ歯類の現像抑制剤をセットアップするとき、前方にジャンプするから動物を保つために必要であるよりも、さらににプランジャーを押さないでください。また、動物に対してプランジャーを押したりしないで、そして時間の長時間制止する人に動物を放置しないでください。 C.尾静脈の可視化軽く軽く、人差し指を介してラップし、親指で指の内側に対して保持されるようにあなたに向かって尾を引く。直立テールのどちらかの側にある尾静脈を識別します。可視化は、その後、エタノールでダウン拭き取り、ぬるま湯で尾を暖めることによって促進される。 D.細胞の注入静脈が位置しているときは、静脈に浅い角度で平行にシリンジのベベル面を上に挿入する。一回静脈内、静かにプランジャーを押し下げて、あなたが静脈にある場合は注射への抵抗はないはずです。何らかの抵抗がある場合は、シリンジを取り外して、別の場所でもう一度試してください。 あなたが静脈内にある場合、注入が完了するまで、ゆっくりとプランジャーを押し下げる。尾は、注入時に膨張になってはいけません。その場合、これは針が静脈内にないことを意味します。注入中に静脈を&quot;失う&quot;ことが可能です。このような場合は、針を取り外して、別の場所で注射をしてみてください。複数の注射をしようとする必要がある場合、それはオリジナルの注射部位からのマウスの体に向かって尾を上下に移動するのが最善です。養子移入におけるもう一つの試みのために自分で部屋を与えるためにテールに遠位に起動して、この可能性のために将来の計画を立てる。 E.ポスト噴射の考慮事項注入が完了した後、静脈からの血液の小さな逆流が発生します。静かに出血が停止するまでクリーンワイプで注射部位を押してください。プランジ​​ャーを外し、ゆっくりと尾部に保持している間、動物は、制止​​する人のうち、前方に歩いてみましょう。 常にあなたが従っているを確認するために、動物のケア委員会および動物使用のプロトコルを確認してください。実際の実験を試みる前に、このテクニックを数回練習することが重要です。 2。リンパ節の分離二光子イメージングのためのリンパ節の分離は、周囲の組織から適切なリンパ節を慎重に除去する必要があります。 A.選択し、末梢リンパ節の位置イメージングのためのリンパ節の選択は、実験目的に依存します。局所感染または射出所属リンパ節に注意してください、とイメージングのための適切なノードを選択します。ヴァンデンブルークらの論文は。、詳細3のマウスリンパ節の位置と形態を説明しています。このビデオでは、我々は細胞の分離やイメージングに適している6大末梢リンパ節の切除を示しています。 B.リンパ節の除去： リンパ節を除去しながら心に組織の整合性を保つ。しない破裂カプセルを、または任意の方法の損傷リンパ節の構造に注意してください。リンパ節の構造的完全性の損失は、実験が損なわれます。必要に応じて、解剖顕微鏡下で解剖細かい（＃5デュモン）ピンセットでリンパ節の表面から脂肪を取り除く。リンパ節は、これは光を散乱によるイメージングを覆い隠すので、ノードの表面上に残りの脂肪を含んではならない。 リンパ節の適切な切除はまた、最初の本当の実験の前に練習する重要な手法です。 3。リンパノッド電子イメージングセットアップと注意事項リンパ節は、この場合には埋め込み式のチャンバーから構成される画像処理の段階、に保護する必要があります。暖め、酸素灌流メディアは、蠕動ポンプとインラインヒーターを使用して1つの側面からチャンバーにポンプでくまれ、そして廃棄物収集フラスコに真空管と収集チャンバ内へ下降傾斜チャネルを介してそれが終了します。私たちのシステムでは、リンパ節は、Cメディアは、37℃で浸漬されており、医療グレードのcarbogen（5％CO 2、95％O 2）と超灌流。可能な限りそのまま灌流媒体の温度は、リンパ節の近くに記録されるべきである。特定のシステムでは、メディアの流量と舞台デザインのバリエーションは、顕微鏡ステージと客観的に対応するために必要となる場合があります。 A.リンパ節イメージングセットアップ： としてビデオに示すように、我々は適切なサイズにカットプラスチックカバースリップに（正立顕微鏡を使用して画像TまたはB細胞のゾーンへの）ダウン髄質側は、最初にそれを添付してリンパ節を固定します。無毒の組織接着剤を（我々は3M社からVetBond™を使用する）使用することが重要です。 カバースリップは、イメージングチャンバーのガラスのベースにこれを付着、カバースリップの下側にシリコーングリースの小さい軽打を配置することによって、流れる媒体に固定されています。 B. 2光子イメージングの考慮事項： いくつかの要因は、リンパ球が動いて停止することができます：高すぎるまたは低すぎる温度、過剰なレーザーパワー、圧縮またはリンパ節へのその他の損傷、及び酸素の欠乏を。過度のレーザパワーや温度による損傷&gt;〜39℃の不可逆的である。 細胞が薄暗いであれば、それはむしろ、細胞を可視化するためにより高いレーザパワーを使用するよりも検出器のゲインまたは細胞標識のプロトコルを増加させるほうがベターです。蛍光タンパク質のラベルまたは式の最適化は、バックグラウンドの自家蛍光のレベル以上の蛍光をもたらすために必要となる場合があります。合理的なゲインおよびレーザパワーの設定では、ベースライン上の蛍光の約2ログのシフト（フローサイトメトリーで測定した）細胞の可視化のための合理的です。 調整可能な二光子レーザーで、励起に使用される波長は、撮像される蛍光体の二重単一光子励起の最大値より少し小さくする必要があります。複数のラベルまたは蛍光タンパク質を用いて二光子実験ではそれは両方のラベルの最適な設定を見つけるために使用される励起波長を実験する必要があるかもしれません。二光子励起は、本質的な青色の第二高調波発生を利用することによって画像内因性のコラーゲンの構造に使用することができます。第二高調波発生（SHG）は、2つの光子が物質内で結合されている場所で発生し、元の光子の2倍の周波数の単一光子を放出する。組織では、二光子励起では入射レーザー光は、コラーゲンのような高度に秩序化されたタンパク質の構造に垂直な場合に発生します。 二光子励起イメージングに関する実務的な問題は、プレビューのレビュー4、5で議論された。

<ol>
	<li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+Cell+Motility+and+Interaction+Dynamics+Imaged+by+Two-Photon+Microscopy+in+Lymphoid+Organs.+Annu+Rev+Immunol.">Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs. Annu Rev Immunol.</a> Annu Rev Immunol. , (2008).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Van den Broeck, W., Derore, A., Simoens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+nomenclature+of+murine+lymph+nodes:+Descriptive+study+and+nomenclatory+standardization+in+BALB/cAnNCrl+mice.">Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice.</a> J Immunol Methods. 312, 12-19 (2006).</li><li>Cahalan, M. D., Parker, I., Wei, S. H., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+tissue+imaging:+seeing+the+immune+system+in+a+fresh+light.">Two-photon tissue imaging: seeing the immune system in a fresh light.</a> Nat Rev Immunol. 2, 872-880 (2002).</li><li>Germain, R. N., Miller, M. J., Dustin, M. L., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+the+immune+system:+progress,+pitfalls+and+promise.">Dynamic imaging of the immune system: progress, pitfalls and promise.</a> Nat Rev Immunol. 6, 497-507 (2006).</li></ol>

このビデオプロトコルでは、養子細胞移入と末梢リンパ節のイメージングリンパ球の運動性に必要なリンパ節の分離と準備するための手順を示しています。二光子イメージングには、外植組織（ここに示されている）の両方でと生体製剤における共焦点イメージングに比べていくつかの利点があります。特に、光散乱を最小限に抑え、リンパ節被膜下イメージング〜300マイクロメートルを可能にするといくつかの組織4の深い赤外励起の使用。また、多光子励起は、漂白写真と焦点面の側面から組織の損傷を最小限に抑え、目標の焦点に制限されています。あらゆる新技術と同様に、しかし、二光子イメージングは、万能薬ではないとその限界と潜在的な落とし穴が念頭5に保持する必要があります。興味のコラーゲン細胞を蛍光外因性のプローブによって、または蛍光タンパク質の発現によってラベル付けされている必要がありますがそのような第二高調波発生などの内因性シグナルを除いて - これらの中でその要件です。真実にそれらがに浸漬されているのに対し、構造要素と無数の他のラベルの付いていない、目に見えない細胞の複雑な環境と相互作用し、印象は、このように、これらの標識細胞が黒色無効に泳いでいることが作成されます。 免疫学への二光子イメージングの導入以来6年で、この手法は、直接細胞間相互作用、細胞の運動性と局在、細胞のシグナル伝達経路と細胞傷害性細胞を含む無傷の生体組織のプロセスで視覚化することによって長年の疑問を解決するための研究を可能にしたイベントを殺す。フィールドは急速に初期の現象論的説明を越えて進化している、とコンピュータモデリングとシミュレーションと一緒に定量分析は、現在、リンパ節内で明らかに混沌とした細胞の動きとの相互作用が効率的な免疫応答を導く方法の意味を理解し始めている。この進展は、総合的にimmunoimagingの2光子顕微鏡のアプリケーションを記述する100以上の論文をリスト最近の出版物1に見直される。

dissection and 2-photon imaging of peripheral lymph nodes in mice

二光子イメージングには、基礎条件の下でリンパ節内および免疫応答1の開始時の運動性と細胞間相互作用の優雅な振り付けを明らかにした。ここで、我々は、標識されたT細胞、リンパ節の分離、およびCD4 + 2002年2で説明したように植リンパ節におけるT細胞のイメージング運動の養子移入するための方法を提示する。免疫細胞の二光子励起イメージングは​​、細胞を蛍光セルトラッカーの色素で染色することによりまたは蛍光タンパク質を発現するかして、ラベル付けされている必要があります。我々は、約15-30分内リンパ器官には、自宅レシピエント動物の尾静脈にドナーマウス由来の細胞を注入するの養子移入の手順を示しています。我々はリンパ節の分離を示し、摘出したリンパ節の実装を適切に確保するための方法を説明します。このような適切な灌流メディアの酸素、温度、およびレーザパワーのような他の考慮事項が説明されています。最後に、我々はナイーブCD4の3D映像+ 37℃で定常状態の運動性を示すT細胞を提示℃に

Watch this Scientific Journal Video about Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice at JoVE.com

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

二光子イメージングには、基礎条件の下でリンパ節内および免疫応答1の間にリンパ球運動と細胞間相互作用を明らかにした。ここで、我々はT細胞、リンパ節の分離、およびCD4 +植リンパ節におけるT細胞のイメージング運動の養子移入を示しています。

解剖とマウスの末梢リンパ節の2 - フォトンイメージング

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

dissection-and-2-photon-imaging-of-peripheral-lymph-nodes-in-mice

Research

JoVE Journal

Biology

46.1K ビュー.  University of California, Irvine (UCI). 二光子イメージングには、基礎条件の下でリンパ節内および免疫応答1の間にリンパ球運動と細胞間相互作用を明らかにした。ここで、我々はT細胞、リンパ節の分離、およびCD4 +植リンパ節におけるT細胞のイメージング運動の養子移入を示しています。 

ビデオ: Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

CD4 +ミルテニーMACS精製を使用してマウスのリンパ節からT細胞の分離

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

生物学

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

2光子顕微鏡を用いたマウスの胸腺のその場イメージングで

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

頭蓋窓を介して新皮質の血流の2光子イメージングのための方法

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

レイヤーin vivoでの2光子カルシウムイメージングマウスの2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

2光子イメージングで使用するための骨髄由来マウス樹状細胞の世代

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

の解剖と染色ショウジョウバエ幼虫卵巣

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

FDG micro-PET/CTマウスにおける褐色脂肪の機能イメージング

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

蛍光タンパク質を発現する生きたマウスでのイメージング細胞内の構造物の生体顕微鏡

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

解剖、イメージング、および薬理学的治療： ショウジョウバエ蛹精巣や独身男性生殖ライン嚢胞のex vivo培養

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

生後マウスにおける聴覚ブラの解剖：中耳の骨の単離および組織学的解析

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...