The authors would like to thank the Biomarker Imaging&#160;Research Laboratory (BIRL) at Sunnybrook Research Institute for their histology services. Support for this work was provided by the Terry Fox Foundation, the Canadian Breast Cancer Foundation&#8208;the Prairie&#8208;NWT as well as a CIHR grant, #MOP-97996.

1。試料<ol><li> 3日間は、授乳の発症を投稿する野生型CDH1から外科的に乳腺だけでなく、IGFBP7欠損マウスを切り出す。 </li><li>ネイティブ乳腺形態を取り戻すためにガラススライド上に腺を広げた。 </li></ol> 2。固定及び組織処理<ol><li> 4 Oで中性緩衝4％PFA O / N中の乳腺を修正しました。 </li><li>組織処理前に70％エタノールでの腺に保管してください。 </li><li>小さな組織処理カセットに腺を転送します。 </li><li>自動化組織プロセッサを使用して組織を処理する<ol><li> 45分間、キシレン中で1時間及び2回、100％エタノール中で、45分間、95％エタノールで45分間、2回、3回、70％エタノールのエタノールおよびキシレン浴を増加させるに組織を脱水する。 </li><li>印加圧力と真空中での1時間ごとに、パラフィン3回組織に浸透。 </li></ol></li><li>ブロックを形成するためにパラフィンに組織を埋め込む、区画する。 </li></ol> 3。組織学およびBlockfaceイメージング<ol><li>過剰のパラフィンが除去されるまでロータリーミクロトームを用いてパラフィンブロックをトリミング。 </li><li>カセットに垂直なパラフィンブロックの少なくとも二つの隅部に1mmの孔を穿孔する垂直フライス盤を使用する。 </li><li>回転式ミクロトームで組織ブロックをマウントします。 </li><li>ミクロトームの前にblockface撮像システム19を設定する。 </li><li>前の切片に光学blockfaceイメージをキャプチャします。 </li><li>ミクロトームで5μmの厚さで4つのセクションでリボンをカット。 <ol><li>冷水浴にリボンを転送します。 </li><li>リボンの第2、第4のセクションを分離し、顕微鏡スライド上にマウントします。第2、第4のセクションを選択すると、セクション間の5μmのギャップを提供します。 </li><li>それは顕微鏡スライド上に再マウントして、それを滑らかにするために、温水浴（48 度C）の各セクションを展開します。 メモ：Cutting、取付部は、涙、倍、収縮、拡張などのセクションにいくつかの歪みを引き起こすunwrinkling。これらの成果は、組織学切片の登録を複雑にする。 </li><li>自動染色装置を使用し、H＆Eでのセクションを染色。 </li><li>自動カバースリッパーを使用してスライドをカバースリップ。 </li><li>興味の解像度でデジタル組織学スライドスキャナを使用してスライドをデジタル化する。このプロトコルの倍率は20倍であり、分解能は0.47μmである。 </li><li> blockface画像の解像度、18μmの組織学的画像をダウンサンプリングする。 </li></ol></li></ol> 4。画像の登録<ol><li>画像分割やポイントの選択<ol><li> blockfaceの画像は、登録孔の画素値を測定し、セグメントパラフィンブロックの角部における整合穴に固定された閾値として平均値を用いる。 </li><li>いくつかの追加部品もによってセグメント化される可能性があるため固定しきい値を使用して、穴を見つけて、余分なオブジェクトを破棄するように真円とセグメント化されたオブジェクトの領域を使用します。これを行うには、小さなコードを書いて、セグメント化されたオブジェクトのための（4πさx面積）/（周長）2の比率を見つける。丸いオブジェクトにこの比率は1である。 </li><li>各乳腺の場合は、基準となる1 blockface画像を選択し、登録ホールやランドマークベースの登録技術の中心を用いて基準にblockface画像の残りの部分を合わせます。 </li><li>整列blockface画像では、手動セグメントや背景から組織を抽出します。プロトコルの残りのためのマスクの中で最もかなりのオブジェクトを使用します。 </li><li> H＆Eのためのセクションでは、自動セグメンテーションのため、以下の手順に従ってください。 <ol><li>背景からセグメント画像に大津しきい値処理技術20を使用し、組織学画像のバイナリマスクを作成。 </li><li> Hを使用して、各マスクの中で最もかなりのオブジェクトを識別し、選択するラベルされたオブジェクトのistogram。 </li><li>組織学およびblockfaceマスクの両方から1ピクセル幅の境界点を抽出します。 </li><li>区分線形フィットのシーケンスで境界点を表現するために、チェーンコードのアルゴリズム21を使用してください。 </li></ol></li></ol></li><li>初期剛体レジストレーション<ol><li>初期剛性のは組織学の境界点とそれに対応するblockface画像間の変換を求めるために、フーリエ記述子アルゴリズム22を使用してください。この最初の変換は、平行移動、回転、スケールファクタが含まれています。 </li><li>当初、各組織学画像を変換前のステップから得た変換。 </li></ol></li><li>剛体レジストレーションの改良<ol><li>ローリングボールフィルター23を使用して、組織学輪郭から高い曲の縁部分を削除します。 </li><li>ランダムに一様分布を使用して残りの組織学の境界点から500点を選びます。 </li><li>ザ·トランスフォームフーリエ記述子から取得した初期変換と組織像のランダムな境界点。 </li><li> blockface境界点のセット全体を選択し、blockface境界ポイント、宛先、および組織学ランダム境界点間の剛体変換を見つけるために反復最近点（ICP）アルゴリズム24を使用しています。 </li><li>前のステップから得整列組織学的画像を変換し、整列した組織学的画像のスタックは、組織学のボリュームを作成します。 </li><li>組織学ボリュームの視覚的なイメージを作成するために、3D可視化ソフトウェアを使用してください。 </li></ol></li><li> 5倍の倍率で画像のスタックを表示する<ol><li>倍率を5倍オリジナルの組織学的画像をダウンサンプリングする。 </li><li>組織学画像の一つで関心領域をトリミング。 </li><li>登録の二つのステップから剛体変換の組合せを使用して、他の5倍の組織学的画像におけるその領域の位置を計算する。 </li><li>レジをトリミング他のすべての組織学的画像で同じサイズの領域への関心のアドオン。 </li><li>最終的に手動で地域間の位置合わせを調整。 2画像を重ね合わせると、他の1以上の画像の1の回転と移動のための値を選択可能にし、その後、アライメントが受け入れられたとき、変換された画像を保存するプログラムを書く。 </li><li> 3D視覚化ソフトウェアを使用して整列5X組織学領域のスタックを表示します。 </li></ol></li></ol>

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The authors have nothing to disclose

本研究では、組織を歪める可能性があり、組織内の内部ランダムに選択されたランドマークまたは移植基準マーカを必要としないシリアル2D組織学的画像から三次元組織学ボリュームを再構成する画像レジストレーションのワークフローを開発した。記載の方法により、光学blockface画像自体は従来セクショニングを参照画像として使用される。我々は、blockface画像を位置合わせするのを助け?...

IGFBP7（結合タンパク質7インスリン様成長因子）はIGF結合タンパク質ファミリーのメンバーであり、IGF1受容体4を結合することが示されている。異種移植片腫瘍モデルにおけるIGFBP7の再導入が大きくアポトーシスおよび細胞老化の誘導を介して7 6腫瘍増殖を阻害しながら、IGFBP7のダウンレギュレーションは、乳癌5における予後不良と相関することが知られている。 IGFPB7の影響を研究するために、IGFBP7ヌルマウスは5（未発表データ）を作成しました。これらのマウスが腫瘍を発症しないが、彼らは卵巣、筋肉および肝臓の組織像の変化だけでなく、乳腺の発達パターニング（未発表データ）の欠陥を示している。欠損マウスは、より小さなゴミの大きさを持っており、複数の大きな同腹仔（未発表データ）を維持することができないように欠陥のある表現型は、最初に示された。 組織学3Dボリュームは、有用なのinformatを提供する可能性を有しているボリュメトリック医用画像中の病理所見の定量および比較分析·評価するためのイオン。三次元共焦点、二光子顕微鏡法は、ローカル·エクステント14における腺の高解像度の細胞形態学的情報を提供することができるが、それは、ビューと深さの限られた視野を有する。組織学のボリュームの再構築がはるかに大きい空間範囲にわたってより多くの情報を提供しています。いくつかの歪みがそのような収縮、拡張、涙、そして折り​​目などの組織切片の調製の間に予想されている伝統的なアプローチを使用。これらの歪みは、それが困難な3次元ボリュームを再構成する3次元スタックにシリアル組織学的画像を登録することを可能にする。欠陥を有する連続したセクションの数は、無傷のセクション間の類似度を増加すると減少し、その結果、登録プロセスがより複雑にされる。 別の方法は、組織切片を登録するために、連続組織学voを作成するために提案されているLUME。いくつかの技術は、強度変動8に依存し、他のものは複数のセクション9の形状に基づいている。いくつかの試験片の解剖学的構造は、ランドマークに基づく登録方法12,13と一緒に目印10,11として使用することができます。しかし、これらの内部構造は、全編を通してず、信頼性の解剖学的構造を特定することはできない、いくつかの試料については検出できない場合があります。いくつかのグループは、ペアワイズ登録アプローチを使用し、連続した組織学的画像を等高線や解剖学的構造16〜18を使用して別のものを登録している。参照画像を使用せずに互いに直列組織学的切片を登録すると、位置合わせ誤差を伝播および組織容積の実際の形状を変更することができる。ペアワイズ登録の方法は、画像のスタック全体で組織学セクションおよび内部構造の形状の一貫性に依存しています。従って、それは、試料の密なサンプリングを必要とする臨床検体については、例えば 、可能性があることは必ずしも可能ではない。 このパイプラインでは、組織学のボリューム再構成19のための参照画像のセットとしてblockface画像を使用しています。 Blockface画像はミクロトームで、各セクションが切断される前に実装した後、パラフィン組織ブロックを撮影している。このように、個々の連続切片カットの損傷が連続切片8,11,15の登録を妨げることはありません。私たちは、他のグループとは別の方法でblockface画像を取り込む。光学ブロック顔画像を光学における規則的なレンズを使用するときに通常発生バレルと遠近歪みを排除又は最小化するためにテレセントリックレンズによって得られる。これは、通常のレンズを用いblockface撮影を行う他の公開された方法、オーバー提案されたアプローチの利点の一つである。画像はTISSの間のコントラスト向上のためのブロックの表面の反射を使用するようにわずかに傾斜した角度で​​撮影されているUEとパラフィン表面とパラフィン表面の下、深さ、組織の影を排除する。写真フィルターはまた、ブロック表面19及びコントラストのバランスをとるために、組織からの光を偏光するために使用される。回転式ミクロトーム上のブロックの位置ずれを補正するために、二から三穴がblockface画像において容易に検出されたブロックの角に掘削されています。これらの穴の重心はblockface画像を位置合わせするためにランドマークベースの剛体レジストレーションと一緒に使用されている。

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reconstruction of 3-dimensional histology volume and its application to study mouse mammary glands

組織学のボリューム再構成は、3D形状および細胞から構成されるマクロ構造のレベルで器官の体積変化の研究を容易にする。また、体積医療画像および療法における新規技術とアルゴリズムを調査し、検証するために使用することができます。異なる器官1,2,3の3D高解像度の地図帳を作成すると、組織学、ボリューム再構成の他のアプリケーションである。これは、組織の構造や様々な細胞機能の間の空間的関係を調査するためのリソースを提供しています。我々は、光blockface画像のセットを使用して組織学ボリューム再構成のための画像登録手法を提示する。再構築された組織学のボリュームには伝播された後処理登録エラーで処理された試料の信頼性の高い形状を表す。 2マウス乳房腺のヘマトキシリン​​およびエオシン（H＆E）染色切片はEDから抽出された境界点を使用して、それに対応するblockface画像に登録されていた組織学およびblockface画像中の試験片のGES。レジストレーションの精度を目視で評価した。乳腺のマクロ構造の位置合わせは、視覚的にも高解像度で評価した。 この研究は、乳腺切除から3次元組織学ボリューム再構成に至るまで、この画像登録パイプラインの異なるステップの輪郭を描く。 2Dの組織学的画像は各項のペア間の構造的な違いを明らかにしながら、3次元組織像ボリュームは形状や乳腺の音量の違いを視覚化する機能を提供します。

従来の顕微鏡技術の落とし穴は、顕微鏡レベルで器官の理解は一度に一つの視野に限定されることである。スライド全体のセクションを提供しても「全開示」スライドを、三次元情報を提供することができない。スライド全体の開発、動的なスキャン技術を使用すると、そのセクション全体を参照する当社の能力が増加している、しかし、構造体を外挿すると、3次元組織学ボリューム再構成...

Watch this Scientific Journal Video about Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands at JoVE.com

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3次元組織学ボリュームとマウス乳腺研究への応用再建

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

reconstruction-3-dimensional-histology-volume-its-application-to

Research

JoVE Journal

Biology

14.3K ビュー.  University of Toronto. We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands. 

ビデオ: Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

タスクを歩くラダーラング：スコアリングシステムとその実用化。

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

レンズゼブラフィッシュでの移植とアイ変異体の解析への応用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

マウス乳腺上皮細胞は乳腺刺激分化の過程でMammospheresを形成し、

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

マウスモデルにおける乳腺の発達と機能の評価

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

マウス乳腺に局在薬物送達のための管内注入

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

初代マウス骨芽細胞から骨細胞培養物を入手するレチノイン酸のアプリケーション

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

腫瘍球の高スループット画像解析：自動的かつ正確にスフェロイドのサイズを測定するための使いやすいソフトウェアアプリケーション

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

マウスで圧力ボリュームループを測定します

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...