我々は、YFPマウスを提供するためのアンソニーJ. Koleske、博士に感謝する。

パート1：マイクロプリズムの光学と顕微鏡のセットアップ<ol><li>我々が使用するmicroprismsはOptosigmaコーポレーション（図1）から購入したBK7ガラス製の1mm、直角プリズムです。 Microprismsは、可能な限り鉗子を使用して処理されます。 </li><li> 2000nmの - マイクロプリズムの斜辺が400から97％以上の反射率を提供するために強化された銀で被覆されている。これはレーザーの励起光と放射される蛍光の両方の内部反射を提供します。 </li><li> Microprismsは常に、可能な場合は鉗子を使用して処理されます。手袋は、プリズムを清潔に保つために、常に着用している。 </li><li>我々は、小動物のイメージングが可能なカスタムビルドの二光子顕微鏡を使用してください。定位装置に接続されている動物は、、3軸電動ステージ上に配置されています。 </li><li>顕微鏡はscanimageソフトウェアを実行しているWindows PCに接続されています。 scanimageでもはPologrutoら1によってMATLABで書かれた画像取得ソフトです。 </li><li>マイクロプリズムのイメージングのために、我々は1024 × 1024または512 × 512ピクセルの解像度で画像を収集する。さらに、我々は通常、2ミリ秒/ラインまたは4ミリ秒/ラインでスキャン。 </li><li>目標の2種類の実験に使用されています： <ul><li>広い視野イメージングのための低開口数（NA）空気の目標（ 試薬および器具の表を参照）。 </li><li> 0で誘発される球面収差を最小限に抑えることのできるガラスの補正環を持つ高NAの空気の目標-ガラスの2mm（ 試薬および器具の表を参照）。 </li></ul></li><li>動物は、低倍率の目標の下でイメージングするための準備が完了すると、マイクロプリズムの上でレーザーの正方形のラスタスキャンパターンを合わせます。これは東洋のイメージを手助けし、レーザパワーの効率的な利用を行います。 </li><li>ガラスの補正環付き高NAの対物レンズを用いて、ガラスの約1mmを補正する補正環を設定します。蛍光画像は、実験中に表示されたら、人は慎重に画像の解像度を最大にするために補正環を最適化するために顕微鏡の内部に到達することができます。 </li></ol>パート2：非生存動物の手術とマイクロプリズムの挿入<ol><li>手順全体の長さは、最初の切開からイメージングが完了するまで、実験の目的によって異なります。完了するまで40分 - しかし、非生存外科手術とマイクロプリズムの配置は、約30を取ることが期待できる。 </li><li>マウス（P30 - P60）が適切に外科的処置に適した平面にケタミン（100 mg / kg体重）及びキシラジン（10 mg / kg）のIP注入を用いて計量し、麻酔されています。 </li><li>実験全体を通して、動物は麻酔のレベルを評価するために15〜20分間隔でチェックする必要があります。動物がしっかりつま先-ピンチに反応する場合、ケタミン/キシラジンの補足投与が必要です。一般的に補足用量は初回投与量の3分の1ですし、必要に応じて即座にIP注射用シリンジに事前に準備しておくことができる。 </li><li>マウスが適切に麻酔したら、動物の頭の毛の大部分は、第40刃とトリマーを使って剃っています。 </li><li> Nairさん®は、撮像中にマイクロプリズムの邪魔になることが毛髪の自由表面を作成するための頭の上に残っている毛に適用されます。 5分後、Nairさん®は綿棒を使用してオフに洗浄される。 </li><li>麻酔動物を適切にマウスの定位装置に配置されます。外科的処置とイメージングのセッション中に動物は摂氏36度に設定された水を充填した加熱パッドに保持されます。 </li><li>適切な場所に固定された動物の頭と、つのクリーンな切開は皮膚を分離するために頭蓋骨の内側行下にメスを用いて行われます。 </li><li> 3％の過酸化水素の少量綿棒に配置され、頭蓋骨と皮膚の間に膜を一掃するために頭蓋骨に適用されます。 </li><li>過酸化水素はまたブレグマ、開頭を行い、マイクロプリズムを挿入する場所を知ることで主要なランドマークを明らかにすることができます。 </li><li>開頭手術が開始される前に、耳のバーやかまバーは適切にそれが表で、基本的にレベルとなるように露出頭蓋骨を傾けるように調整されています。これは、開頭術のための優れたプラットフォームを提供します。さらに、これは入ってくる励起光をプリズムに直交の上面になります。これは撮影しながら光学収差を最小限に抑えることができます。 </li><li>小さな歯のバリは（〜0.8 mmヘッドサイズ）フレキシブルエクステンションアームを使用してドレメル®ツールに接続されています。 craniotomiesの場合、我々は、約7,000〜10,000 rpmの速度を設定します。 </li><li>正方形の溝が骨に確立されるまで、歯科用バーは、頭蓋骨に沿ってざっとれる。正方形の辺の長さは約2〜3 mmであると1.5mmの尾とブレグマへ横1 mmの中心。 </li><li>小さな開口部が頭蓋骨を介して行われるまで、骨を薄く継続。鉗子CAのペアnは離れて頭蓋骨から骨弁を持ち上げる。 </li><li>硬膜は、マイクロプリズムが皮質に挿入する前に削除する必要があります。出血は、最高の血液は、数分間表面に凝固させることによって制御されます。その後、凝固血液はマイクロプリズムを挿入する前に手術用ス​​ポンジを使用して削除されます。 </li><li>皮質とプリズムの配置を支援する支援するために、マイクロプリズムの垂直面は、鉗子のハンドルと平行にラインアップしています。 </li><li>プリズムの上部は新皮質の表面と同一平面になるまで適切に開催されたプリズムと、全体のマイクロプリズムが挿入されます。 </li><li>正しく挿入されると、マイクロプリズムは、適切な位置に残ります。ということに起因するいかなる出血は最小限で、小さな手術用スポンジで吸収することができる。プリズムが配置されると動物は、イメージングのための準備ができています。 </li><li>マイクロプリズムは動物で使用された後、それは何度も再使用することができます。しかし、それは適切に残っている生物学的物質を除去するクリーニングする必要がありません。これをメタノールに浸漬し、次に塩酸酸の小さなボリュームにプリズムを浸漬することによって行うことができます。洗浄マイクロプリズムは、将来の使用まで、レンズペーパーでラップできます。 </li></ol>パート3：代表的な結果<img src="/files/ftp_upload/1509/1509fig1.jpg" alt="図1" /> 図1：右アングルマイクロプリズムの画像。この実験で使用したプリズムは、励起および発光光の内部反射を可能にするために斜辺上で強化された銀のコーティングとの間に1ミリメートル、BK7ガラスのプリズムです。 <img src="/files/ftp_upload/1509/1509fig2.jpg" alt="図2" /> 図2：レイヤーV YFP錐体細胞のイメージ。皮質表面から深さ900μmの - マイクロプリズム付きワイド視野イメージングは​​、錐体細胞体〜800の大規模な人口を明らかにする。さらに、層に向かって伸びる尖頭の樹状突起は、私も解決することができます。スケールバー= 200μmの。 <img src="/files/ftp_upload/1509/1509fig3.jpg" alt="図3" /> 図3：マイクロプリズムと組み合わせて使用するより高い開口数の目標は、V層錐体ニューロンで尖頭の樹状突起上の樹状突起を解決するために十分な空間分解能を提供します。スケールバー=10μmである。 <img src="/files/ftp_upload/1509/1509fig4.jpg" alt="図4" /> 図4：マイクロプリズムと蛍光色素の尾静脈注射を用いて得られた画像。画像は、皮質のボリュームを介して分岐深い脳の領域と小さな毛細血管から伸びる縦、大口径の血管を示しています。スケールバー= 200μmの。 <img src="/files/ftp_upload/1509/1509fig5.jpg" alt="図5" /> 図5：高開口数の対物レンズを用いて、新皮質の毛細血管のネットワークは、より詳細で画像化することができます。スケールバー= 50μmの。 <img src="/files/ftp_upload/1509/1509fig6.jpg" alt="図6" /> 図6：Microprismsはまた血管を流れるイメージング赤の血液細胞（赤血球）が可能になります。赤血球は蛍光色素を吸収しないため、容器全体の暗い縞と見られている。容器全体のラインスキャンによって、人はRBCフラックスと速度の測定値を得ることができます。スケールバー=10μmの（上部と下部の画像で水平バー）と250ミリ（下の画像の縦棒）。

<ol>
	<li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomed Eng Online. 2, 13-13 (2003).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28 (1), 41-41 (2000).</li><li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex.">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> Proc Natl Acad Sci U S A. 95 (26), 15741-15741 (1998).</li></ol>

動物実験はエール大学のIACUCによって定められたガイドラインおよび規制に準拠して行った。

イメージングの実験ではマイクロプリズムを使用しては比較的簡単ですし、新皮質上でin vivo試験のための多くの利点を提供しています。このテクニックを使用する代表的な例としては、V層の皮質ニューロンにおける黄色蛍光タンパク質を発現するトランスジェニックマウスから撮影した画像が含まれています。 microprismsを使用して、一つはV層、皮質表面の下に約1 mmの大錐体細胞体のコレク...

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<td>With enhanced silver reflective coating-(R77)</td>
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<td>0.28 NA, 4x air objective</td>
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<td>0.60 NA, 40x air objective</td>
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in vivo imaging of deep cortical layers using a microprism

我々はマイクロプリズムの形状で深脳イメージングプローブを用いて皮質組織のin vivoイメージングのためのプロトコルを提示する。 Microprismsのサイズは1 mmのものであり、励起および発光光の内部反射を可能にするために斜辺上に反射コーティングを持っている。マイクロプリズムは通常、スライス標本に見られるような視点で同時に複数の皮質層の画像を調査。画像は、大視野（〜900μmの）で収集されます。さらに、我々は非生存外科手術と顕微鏡のセットアップに関する詳細を提供します。代表的な結果は、表面的な皮質層を貫通して延びると房に延び、その尖頭の樹状突起を示す汝- 1 YFP - HマウスからV層錐体ニューロンの画像が含まれています。分解能はV層のニューロンの細胞体の近くに画像の樹状突起棘に十分であった。蛍光色素の尾静脈注射は、皮質の血管の複雑なネットワークを明らかにする。毛細血管を流れる赤血球（RBC）のラインスキャンは、赤血球速度とフラックスレートが得られることを明らかにする。この小説マイクロプリズムのプローブは、深い細胞構造とin vivoでの皮質機能の可視化のエレガント、かつ強力な新しい方法です。

Watch this Scientific Journal Video about In vivo Imaging of Deep Cortical Layers using a Microprism at JoVE.com

In vivo Imaging of Deep Cortical Layers using a Microprism

マウスの新皮質に挿入直角microprismsは通常、スライスに見られる視点を持つ複数の皮質層の深いイメージングが可能になります。一ミリmicroprismsは、広い視野（〜900μm）と樹状突起棘を解決するために十分な空間解像度を提供します。我々はV層神経イメージングとmicroprismsを使用して新皮質の血管イメージングを示す。

マイクロプリズムを使ってDeep皮質層のin vivoイメージング

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

in-vivo-imaging-of-deep-cortical-layers-using-a-microprism

Research

JoVE Journal

Biology

In vivo Imaging of Deep Cortical Layers using a Microprism

11.3K ビュー.  Yale University. マウスの新皮質に挿入直角microprismsは通常、スライスに見られる視点を持つ複数の皮質層の深いイメージングが可能になります。一ミリmicroprismsは、広い視野（〜900μm）と樹状突起棘を解決するために十分な空間解像度を提供します。我々はV層神経イメージングとmicroprismsを使用して新皮質の血管イメージングを示す。

ビデオ: In vivo Imaging of Deep Cortical Layers using a Microprism

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

解離マウス皮質ニューロンの培養の準備

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

生物学

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

AMフラ-2を用いて皮質ニューロンのカルシウムイメージング

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

IVISスペクトラムを用いた乳腺腫瘍の in vivo生物発光イメージングに

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

ショウジョウバエの胚の生体内の遠心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

イメージングタンパク質間相互作用 in vivoで</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

遺伝子調節の1分子蛍光イメージング<emインビボ&gt;</em&gt;開裂により同時翻訳アクティベーションの使用（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

多重化された免疫組織化学染色およびマルチスペクトルイメージングを用いたタンパク質発現と共局在の定量

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

の剛性を測定します<em&gt; ex vivoで</em原子間力顕微鏡を使用&gt;マウス大動脈

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...