NCI（U54 CA00100970）、NCI（1 R03 CA150040 - 01）、ACS（116602 - PF - 09 - 018 - 01 - CNE）

1。腹部乳腺の除去と全山の準備<ol><li> IACUCガイドラインに従って動物を安楽死させる。表面の板の上に針、またはテープの足でその足から背中に動物を固定し、エタノール（EtOH）でぬれた皮膚を拭いてください。皮膚を鉗子で少し持ち上げて、鋭いはさみが首に向かってペア＃5の乳首とカットの間に始まる皮膚に正中切開を行うことで。後脚に向かって正中線からの反転Yの切開を行います。胸部乳腺を収集することもある場合は、＃2乳首前足に向かってペア間の正中線からもYの切開を行います。 </li><li>腹部の第4乳腺を公開するために逆Yの切開の片側にハサミで開いている肌を細かく分析、乳腺、皮膚に添付されています。完全に腺を露出させる、表面の基板への針で皮膚をピン。一度に片側と腺を働く。 </li><li>近くに乳頭に近位領域から開始し、動物の背骨に向かって腺の遠位端に向かって作業を、鋭いはさみおよび/またはメスを使ってどちらかの皮膚からフリー乳腺を解剖。 （これは特に高齢ラットでは、数分かかる場合があります。） </li><li>すぐに適切にラベルをガラススライド（&quot;ダイヤモンドのペン&quot;適切な永続的なマーカーのペンと/またはを使用する）に分離された腺を広げ、そしてその場で元のサイズと形状に慎重に対応する腺を広げる。スライドガラスは、腺よりも大きい必要があります。スライドに腺を拡散した後、腺がスライド上に付着し、それを乾燥させないように、それはしばらくの間、テーブルの上に座ってみましょう。 </li><li>カルノア固定液（75％氷酢酸、25％の無水エタノールを=エタノール）を含むjarファイルにスライドを入れ、それが2日以上のためのドラフト内で室温（RT）に固定させる。スライドはまた時間のより長い期間のための固定液にしておくことができます。 </li><li>室温で1時間、70％EtOH中でスライドを洗浄します。 </li><li>室温で30分間蒸留水ですすいでください。 </li><li>スライドの裏面を見て）、（あなたがリンパ節が通過染色されていることが表示されるまで2日間以上）*を染色カルミネ明礬で染色。 </li><li> - &gt; 95％ - &gt; 100％70％：エタノール、それぞれの1時間の増大シリーズで洗ってください。無水EtOHで最後の洗浄の後、キシレンで腺を置く。少なくとも2日間室温でドラフトに座ってみましょう。この最後のステップでは、乳房脂肪パッドの乳腺の意味の脱脂をクリアし、透明性のそれに続く増加しています。 fattier腺はより長いクリア時間が必要な場合があります。 </li><li>このようなPermountとしてマウントメディアを、使用してカバースリップをマウントします。実体顕微鏡下で観察する前に（数日）もスライド乾かします。 *カルミネ明礬は染色： 場所1 500 mLの蒸留水と20分間沸騰のgカルミン（シグマC1022）と2.5gの硫酸アルミニウムカリウム（シグマA7167）。水で500 mLに、最終的な音量を調整します。フィルタリングと冷蔵。ソリューションは、数ヶ月のために使用することができます。色が弱くなったときに捨ててください。 </li></ol> 2。乳腺全山のモルフォロジーの解析乳腺の全体のマウントの形態は、以下のエンドポイントと乳癌リスクとの相関結果に基づいて分析されます。 <ol><li>生後21日で、乳腺のホールマウント（思春期前の年齢）： <ul><li>上皮成長： <ul><li>定規を用いて測定した乳首から上皮木の端（単位：mm）までの距離、 </li></ul></li><li>悪性転換の可能性： <ul><li> TEBsの数（末端の芽）、光学顕微鏡下で数えた。 TEBsは乳腺上皮ツリーの先端に位置する最大の球根状の構造であり、 </li></ul></li><li>分化： <ul><li> AB1（肺芽）スコア（0-5）。歯槽芽が上皮に分散されています。 </li><li> AB2スコア（0-5） </li></ul></li></ul> ホールズのマウントは、光学顕微鏡下で採点されます。 AB1およびAB2からスコアの値は、最終分化のスコアに追加されています。 </li><li>乳腺は、ホールマウントPND 50（後の思春期の年齢）で収集： <ul><li>上皮成長： <ul><li>リンパ節から上皮木の端（単位：mm）までの距離は、定規を用いて測定。 </li><li>上皮ツリーの先端からの脂肪パッドの端までの距離は、定規を用いて測定。 </li></ul></li><li>悪性転換の可能性： <ul><li>光学顕微鏡下でカウントTEBsの数、 </li></ul></li><li>分化： <ul><li> AB1スコア（0-5） </li><li> AB2スコア（0-5） </li><li>小葉のスコア（0-5） </li></ul></li></ul> ホールズのマウントは、光学顕微鏡下で採点されます。差別化は、2つの方法で評価することになります。最初に、AB1、AB2および小葉からスコアの値は、最終分化のスコアに追加されています。さらに、小葉のスコアとAB1 + AB2のスコアとの比率が計算されます。 ABSは小葉に分化する、と小葉とABSとの間の比率が高いほど、より分化腺があります。 </li></ol> 3。触診とラットと乳腺の形態との相関性の乳腺腫瘍の測定<ol><li>この手順を開始するには、頭の後ろで人差し指と反対側の腋窩の下に親指と中指で、戻って上の手のひらで全身を把握し、ラットを保持する。それは、その後部の上に横たわっているように、ラットを回し、任意の乳腺腫瘍を検出するために触診、触知腫瘍が&quot;塊&quot;のように感じるはず。 </li><li>次の幅、長さ、および腫瘍の高さを測定するためにキャリパーを使用。楕円の式を使用して腫瘍体積を計算する、ボリューム=1/6πabc、&#39;A&#39;=幅、&#39;B&#39;=長さ、および&#39;c&#39;=高さ。 </li><li>手動で週単位でノートブックの腫瘍の位置とサイズを記録。このデータは、後でスプレッドシートに転送されます。 </li></ol> 4。代表的な結果乳腺脂肪体とwholemountにその処理を慎重に解剖は乳腺の発達状態の評価が可能になります。各ステップが正しく完了すると、全体の乳腺上皮ツリーは、明らかに脂肪パッド内に表示されており、これはTEBsと肺芽や小葉の密度の計算の数を容易に決定することができます。準備wholemountsの失策は、全体の脂肪パッド、不十分な固定、および不十分なクリアを解明するため、障害が含まれる場合があります。 乳腺の形態の評価は、乳腺腫瘍（TEBs）、上皮構造（歯槽の芽と小葉）の分化の程度、および成長の尺度（管伸長）を生じさせることができる構造の現在の数に関する情報を提供します。後者は、上皮の遠位端に配置されて、同様にTEBsに、彼らはTEBsより小さく、悪性腫瘍を生じさせることはありません。それは、ターミナルの端にTEBsを分離することが重要です。ターミナルの端は、上皮ツリー内で見られている。 <img src="/files/ftp_upload/2260/2260fig1.jpg" alt="図1" /> 図1：よく準備乳腺全体のマウントの代表的な結果は、腺のすべてのコンポーネントが適切に解剖されており、カルミン染色は最適です。 <img src="/files/ftp_upload/2260/2260fig2.jpg" alt="図2" /> 図2：不十分な準備乳腺全体のマウントの代表的な結果は腺が正しく適切に引き伸ばされていないと脱脂が完全に発生していない、（腺の先端部分が欠落している）解剖されています。

<ol>
	<li>Rasmussen, S. B., Young, L. J. T., Smith, G. H., Ip, M. M., Asch, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+mammary+gland+whole+mounts+form+mice.">Preparing mammary gland whole mounts form mice.</a> Methods in Mammary Gland Biology and Breast Cancer Research. , 75-85 (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+statements+from+Mammary+Gland+Whole+Mount+Round+Robin+meeting.">Consensus statements from Mammary Gland Whole Mount Round Robin meeting.</a> , (2009).</li><li>Hilakivi-Clarke, L., Clarke, R., Onojafe, I., Raygada, M., Cho, E., &amp;amp, L. i. p. p. m. a. n., E, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+maternal+diet+high+in+n-6+polyunsaturated+fats+alters+mammary+gland+development,+puberty+onset,+and+breast+cancer+risk+among+female+rat+offspring.">A maternal diet high in n-6 polyunsaturated fats alters mammary gland development, puberty onset, and breast cancer risk among female rat offspring.</a> Proc Natl Acad Sci. 94, 9372-9377 (1997).</li><li>Russo, I. H., Russo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+neoplasia+in+long-term+rodent+studies.">Mammary gland neoplasia in long-term rodent studies.</a> Environ. Health Perspect. 104 (9), 938-967 (1996).</li><li>Hilakivi-Clarke, L., de Assis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+origins+of+breast+cancer.">Fetal origins of breast cancer.</a> TRENDS in Endocrinol and Metabol. 17 (9), 340-348 (2006).</li><li>Hilakivi-Clarke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+modulation+of+terminal+end+buds:+its+relevance+to+breast+cancer+prevention.">Nutritional modulation of terminal end buds: its relevance to breast cancer prevention.</a> Curr Cancer Drug Targets. 7 (4), 465-474 (2007).</li><li>Silva Idos, S., De Stavola, B., Mccormack, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+Group+On+Pre-Natal+Risk+Factors+And+Subsequent+Risk+Of+Breast+Cancer.+Birth+size+and+breast+cancer+risk:+re-analysis+of+individual+participant+data+from+32+studies.">Collaborative Group On Pre-Natal Risk Factors And Subsequent Risk Of Breast Cancer. Birth size and breast cancer risk: re-analysis of individual participant data from 32 studies.</a> PloS Medicine. 5, e193-e193 (2008).</li><li>Warri, A., Saarinen, N. M., Makela, S., Hilakivi-Clarke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+early+life+genistein+exposures+in+modifying+breast+cancer+risk.">The role of early life genistein exposures in modifying breast cancer risk.</a> Br J Cancer. 98, 1485-1493 (2008).</li><li>Russo, J., Hu, Y. F., Yang, X., Russo, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental,+cellular,+and+molecular+basis+of+human+breast+cancer.">Developmental, cellular, and molecular basis of human breast cancer.</a> J Natl Cancer Inst Monogr. , 17-37 (2000).</li><li>Oza, A. M., Boyd, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammographic+parenchymal+patterns:+A+marker+of+breast+cancer+risk.">Mammographic parenchymal patterns: A marker of breast cancer risk.</a> Epidemiological Reviews. 15, 196-208 (1993).</li></ol>

利害の衝突は宣言されません。

乳腺の形態学的エンドポイントおよび上皮木の成長の評価は、初期の生活食事の操作、または子宮内あるいは思春期前のホルモン環境に変化させる他の操作は、後で乳癌のリスクを変更するかどうかを予測するために使用することができます。ヒトの乳がんは、ラットにおけるTEBsに似て乳腺構造（端子乳管小葉単位、TDLUs）で開始されるため、この手法は乳癌リスクに影響を与える初期生活の暴露の可能性を決定するために使用することができます。さらに、女性に高いマンモグラフィ密度は4〜6倍に乳癌のリスクを増加し、乳腺上皮木の成長の評価は、マンモグラフィ濃度とこの密度を下げる要因を決定する要因を特定するために使用することができます。この情報を取得するには、乳腺が完全に、解剖し適切にそのすべてのコンポーネントは、顕微鏡下で可視化できるように、スライドガラス上に引き伸ばすことがあります。さらに、適切なカルミン染色とキシレンに脱脂は、乳癌のリスク評価に適している全体のマウントを提供します。

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Glacial acetic acid</td>
<td>&nbsp;</td>
<td>EM</td>
<td>AX0073-9</td>
<td>Carnoy's Fixative</td>
</tr>
<tr>
<td>Absolute ethanol</td>
<td>&nbsp;</td>
<td>The Waner-Graham Co.</td>
<td>64-17-5</td>
<td>Carnoy's Fixative</td>
</tr>
<tr>
<td>Carmine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1022</td>
<td>Carmine solution</td>
</tr>
<tr>
<td>Aluminum potassium sulfate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7167</td>
<td>Carmine solution</td>
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changes in mammary gland morphology and breast cancer risk in rats

子宮と思春期の時の食事/ホルモン因子へのエクスポージャーその乳がんショーの齧歯類モデルでの研究は、乳腺が広範なモデリングとリモデリングを受けるとき、発癌物質誘発性乳腺腫瘍の感受性を変化させる。同様の知見がヒトに記載されている：例えば、高い出生時体重は、乳がんの発症後のリスク増大、および小児期の大豆の摂取は乳癌リスクを減少させる。それはこれらの出生前および出生後の食事の変更が順番に人生の後半で乳がんのリスクを変更することを乳腺の永続的な形態変化を誘導すると考えられている。これらの形態学的変化は、おそらくそのようなDNAメチル化の変化、ヒストンし、遺伝子の転写に影響を与えるmiRNAの発現などのエピジェネティックな変更を、反映している。この記事では、乳腺の形態の変化がラットで乳腺がんのリスクを予測する方法について説明します。我々のプロトコルは、特に細かく分析し、除去ラット腹部乳腺を、どのように乳腺全体のマウントを準備する方法について説明します。また、3つのエンドポイント（終端蕾の数、上皮伸長と分化）することと乳腺癌を発症するリスクを予測するためにデータを使用するに応じて乳腺の形態を分析する方法について説明します。

Watch this Scientific Journal Video about Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats at JoVE.com

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

我々のプロトコルは、ラット腹部乳腺を細かく分析し、どのように乳腺全体のマウントを準備する方法について説明します。また、3つのエンドポイント（終端蕾の数、上皮伸長と分化）を使用して、乳腺の形態を分析し、食事の修飾にさらされたラットで乳腺がんのリスクを予測するためにこれらの結果を使用する方法について説明します。<em&gt;子宮内で</em&gt;または思春期前の時期の間に。

ラットにおける乳腺の形態と乳癌リスクの変化

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

changes-in-mammary-gland-morphology-and-breast-cancer-risk-in-rats

Research

JoVE Journal

Biology

29.3K ビュー.  Georgetown University. 我々のプロトコルは、ラット腹部乳腺を細かく分析し、どのように乳腺全体のマウントを準備する方法について説明します。また、3つのエンドポイント（終端蕾の数、上皮伸長と分化）を使用して、乳腺の形態を分析し、食事の修飾にさらされたラットで乳腺がんのリスクを予測するためにこれらの結果を使用する方法について説明します。子宮内...

ビデオ: Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

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 ...

生物学

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

シングルおよび多細胞コンテキストでInvadopodia媒介細胞外マトリックスタンパク分解の定量測定

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

三次元蛍光画像から細胞形態の変化を定量分析ツール

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

3D原子力組織の変化を研究するために3D保存状態間期核に組み合わさ免疫やDNA FISH

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

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 ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

マウス乳腺の凍結切片の間接免疫

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

乳癌細胞株における細胞増殖を決定するための3つの異なる方法の比較

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

そのままに、全体のマウス乳腺の腺の形態と細胞外マトリックス式の分析のための分離

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

乳腺生検する方法

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

交換可能な蓋付き乳房イメージングウィンドウを使用した縦方向のバイタル内顕微鏡

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...