著者らは、この方法とTiago Ferreira博士（McGill University、カナダ、ケベック州モントリオール）の継続的な成功のために、Michael Islingling博士（ノースカロライナ州ダーラム）を認めたいと思っています。 Shollアプリケーションのサポート。

<ol>
	<li>Sholl, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+organization+of+the+neurons+in+the+visual+and+motor+cortices+of+the+cat.">Dendritic organization of the neurons in the visual and motor cortices of the cat.</a> J Anat. 87 (4), 387-406 (1953).</li><li>Ferreira, T. A., Iacono, L. L., Gross, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+receptor+1A+modulates+actin+dynamics+and+restricts+dendritic+growth+in+hippocampal+neurons.">Serotonin receptor 1A modulates actin dynamics and restricts dendritic growth in hippocampal neurons.</a> Eur J Neurosci. 32 (1), 18-26 (2010).</li><li>Stanko, J. P., Easterling, M. R., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Sholl+analysis+to+quantify+changes+in+growth+and+development+in+rat+mammary+gland+whole+mounts.">Application of Sholl analysis to quantify changes in growth and development in rat mammary gland whole mounts.</a> Reprod Toxicol. 54, 129-135 (2015).</li><li>Assis, S., Warri, A., Cruz, M. I., 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=Changes+in+Mammary+Gland+Morphology+and+Breast+Cancer+Risk+in+Rats.">Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats.</a> J. Vis. Exp. (44), e2260 (2010).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Mammary+Gland+Development+and+Function+in+Mouse+Models.">Evaluation of Mammary Gland Development and Function in Mouse Models.</a> J. Vis. Exp. (53), e2828 (2011).</li><li>ImageJ User Guide. IJ 1.46r. ImageJ Available from: <a target="_blank" href="https://imagej.nih.gov/ij/docs/guide/146.html">https://imagej.nih.gov/ij/docs/guide/146.html</a> (2012)</li><li>Tucker, D. K., Foley, J. F., Hayes-Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+High-quality+Hematoxylin+and+Eosin-stained+Sections+from+Rodent+Mammary+Gland+Whole+Mounts+for+Histopathologic+Review.">Preparation of High-quality Hematoxylin and Eosin-stained Sections from Rodent Mammary Gland Whole Mounts for Histopathologic Review.</a> Toxicol Pathol. 44 (7), 1059-1064 (2016).</li><li>Fenton, S. E., Hamm, J. T., Birnbaum, L. S., Youngblood, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+abnormalities+in+the+rat+mammary+gland+following+gestational+and+lactational+exposure+to+2,3,7,8-tetrachlorodibenzo-p-dioxin+(TCDD).">Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).</a> Toxicol Sci. 67 (1), 63-74 (2002).</li><li>Murray, T. J., Maffini, M. V., Ucci, A. A., Sonnenschein, C., Soto, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+gland+ductal+hyperplasias+and+carcinoma+in+situ+following+fetal+bisphenol+A+exposure.">Induction of mammary gland ductal hyperplasias and carcinoma in situ following fetal bisphenol A exposure.</a> Reprod Toxicol. 23 (3), 383-390 (2007).</li><li>Moral, R., Santucci-Pereira, J., Wang, R., Russo, I. H., Lamartiniere, C. A., 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=In+utero+exposure+to+butyl+benzyl+phthalate+induces+modifications+in+the+morphology+and+the+gene+expression+profile+of+the+mammary+gland:+an+experimental+study+in+rats.">In utero exposure to butyl benzyl phthalate induces modifications in the morphology and the gene expression profile of the mammary gland: an experimental study in rats.</a> Environ Health. 10 (1), 5 (2011).</li><li>Johnson, M. D., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+dimensional+multiphoton+imaging+of+fresh+and+whole+mount+developing+mouse+mammary+glands.">Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands.</a> BMC Cancer. 13, 373 (2013).</li><li>Enoch, R. R., Stanko, J. P., Greiner, S. N., Youngblood, G. L., Rayner, J. L., Fenton, S. E. <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+development+as+a+sensitive+end+point+after+acute+prenatal+exposure+to+an+atrazine+metabolite+mixture+in+female+Long-Evans+rats.">Mammary gland development as a sensitive end point after acute prenatal exposure to an atrazine metabolite mixture in female Long-Evans rats.</a> Environ Health Persp. 115 (4), 541-547 (2007).</li><li>Hovey, R. C., Coder, P. S., Wolf, J. C., Sielken, R. L., Tisdel, M. O., Breckenridge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+mammary+gland+development+in+female+Long+Evans+rats+following+in+utero+exposure+to+atrazine.">Quantitative assessment of mammary gland development in female Long Evans rats following in utero exposure to atrazine.</a> Toxicol Sci. 119 (2), 380-390 (2011).</li><li>Mandrup, K. R., Hass, U., Christiansen, S., Boberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinatal+ethinyl+oestradiol+alters+mammary+gland+development+in+male+and+female+Wistar+rats.">Perinatal ethinyl oestradiol alters mammary gland development in male and female Wistar rats.</a> Inter J of Androl. 35 (3), 385-396 (2012).</li></ol>

著者は何も開示することはない。

出産から思春期まで、乳腺の成長はアロマティックである。思春期後、乳腺は、乳腺上皮が脂肪パッド全体を占有するまで、広範な管分岐および伸長を経て発達する。分枝特性は乳腺発達の重要な側面であり、これらの特性を客観的に定量化する能力は、乳房の正常な発育を評価し、乳房毒性物質への早期曝露後の異常な発達を同定するために非常に有用であり得る。 ?...

乳腺分枝は、一般に腺発生の指標として評価される特性であるが、客観的に定量化することは困難である。 1953年にSholl 1は、猫の視覚および運動皮質におけるニューロンの樹状突起形成を測定する方法を記載し、この技術のためのプラグインはFerriera らによって開発された2 。ニューロンと乳腺の両方が類似の樹状構造を示すため、このプラグインを用いて、周辺期ラット乳腺の2D画像における乳房上皮分岐密度を定量化した。周産期は、離乳は生涯の段階であるため、分析のために選択されました。これは、多くの場合、学術機関や試験ガイドラインの研究で評価されています。 Sholl分析は、FIJIと共に配布されているプラ​​グインです。これはオープンソースの画像処理パッケージImageJで、追加のプラグインが含まれています。プラグインはpredefを囲む一連の同心リングを作成します（典型的には、ニューロンの体細胞または乳腺の主ダクトの起源）を対象とし、対象物の最遠位部分（包囲半径）まで伸びる。次に、各リング上に発生する交差点の数（N）を数えます。また、プラグインはSholl回帰係数（ k ）を返します。これは上皮分岐の減衰率の測定値です。 ImageJを使用して、乳腺全身の骨格化画像が作成され、乳房上皮領域（MEA）が測定される。 Sholl分析プラグインを使用して画像が分析され、ここでは利用されない他の値のうちNとkの値が返されます。乳腺上皮分枝密度は、N / MEAを計算することによって決定される。腺上皮の外側領域で分枝が継続する程度は分枝の複雑さであり、均一な遠位上皮成長の指標である。 kはエピソードにおける遠位減少の尺度であるのでこれは分枝の複雑さと乳房発達の信頼できる指標の有効な尺度である。 このプロトコルは、乳腺全身マウントの骨格化画像を作成し、周辺性雄性および雌性ラットにおける乳房分枝特性を定量的に評価するコンピュータ支援法を記載する。この方法は比較的迅速であり、特別な顕微鏡検査装置の使用を必要としない。この方法の開発および検証はStanko et al。 （2015） 3 。この報告書はまた、ラット乳腺全体=マウントの調製を記載している。同様の哺乳類全体マウント手順が、アシス（Assis） らに記載されている。 （2010） 4およびPlante et al。 （2011） 5 。

<table border="0" cellpadding="0" cellspacing="0" width="1069">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Dissecting board</td>
			<td style="width:161px;">NA</td>
			<td style="width:224px;">NA</td>
			<td style="width:439px;">A piece of styrofoam roughly 10&#34;x12&#34; is suitable.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Dissecting T-Pins</td>
			<td>Daigger</td>
			<td style="width:224px;">EF7419A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Spray bottle with ethanol</td>
			<td>NA</td>
			<td style="width:224px;">NA</td>
			<td>70% ethanol is suitable.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Curved dissecting scissors</td>
			<td>Fine Science Tools</td>
			<td style="width:224px;">14569-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Straight dissecing scissors</td>
			<td>Fine Science Tools</td>
			<td style="width:224px;">14568-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Curved forceps</td>
			<td>Fine Science Tools</td>
			<td style="width:224px;">11003-12</td>
			<td></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:245px;">Superfrost Plus 24x75x1 mm microscope slides</td>
			<td>ThermoFisher Scientific</td>
			<td>4951PLUS-001</td>
			<td style="width:439px;">Thermo Scientific Superfrost Plus &#38; Colorfrost Plus slides hold tissue sections on permanently without the need for expensive coatings in IHC and Anatomical Pathology applications. This treatment reduces tissue loss during staining as well as hours of slide preparation. Slides electro-statically attract frozen tissue sections and cytology preparations and feature a chemistry similar to silane, although optimized to improve application performance. 
			https://www.thermofisher.com/order/catalog/product/4951PLUS4.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Fisherfinest Premium Cover Glass 24x60x1 mm</td>
			<td>Fisher scientific</td>
			<td style="width:224px;">12-548-5P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Bemis Parafilm M Laboratory Wrapping Film</td>
			<td>Fisher scientific</td>
			<td style="width:224px;">13-374-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A9967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethanol absolute, &#8805;99.8% (GC)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:224px;">24102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carmine alum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C1022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum potassium sulfate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Permount mounting media</td>
			<td>Fisher Scientific</td>
			<td style="width:224px;">SP15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Macroscope</td>
			<td>Leica</td>
			<td>Z16 APO&#160;</td>
			<td rowspan="3" style="width:439px;">This is the image capturing hardware and software used in this laboratory.&#160; As there are many different options, the methods and applications may vary between laboratories.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Digital camera</td>
			<td>Leica</td>
			<td style="width:224px;">DFC295</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Camera software</td>
			<td>Leica</td>
			<td style="width:224px;">Leica Application Suite v3.1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">ImageJ software</td>
			<td>Open source</td>
			<td>http://imagej.net/Welcome</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sholl analysis&#160;</td>
			<td>Open source</td>
			<td>http://imagej.net/Sholl_Analysis</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying branching density in rat mammary gland whole-mounts using the sholl analysis method

げっ歯類の乳腺を化学暴露の発生毒性を評価するためのエンドポイントとして利用する研究が増えています。これらの曝露が乳腺発達に及ぼす影響は、典型的には、基本的な寸法測定または形態学的特徴の採点によって評価される。しかし、発達の変化を解釈するための幅広い方法は、研究所間で一貫性のない翻訳につながる可能性があります。研究を通して比較されるデータから適切な解釈が形成されるように、一般的な評価方法が必要です。本研究は、乳腺の分岐特性を定量化するSholl分析法の応用を記載している。 Sholl法はもともとニューロンの樹状パターンの定量化に使用するために開発されました。オープンソースの画像解析ソフトウェアパッケージであるImageJと、この解析用に開発されたプラグインを使用することにより、乳腺分枝密度とam周産期の雌ラットの臍帯腺を決定した。ここに記載されている方法は、Sholl分析を乳腺発達の重要な特徴を定量化するための有効なツールとして使用することを可能にする。

このプロトコールで分析した乳腺の測定された封入半径、MEA、N、 kおよび計算された分枝密度の値を表1に報告する。 Sholl解析では、各半径（ 図9 ）および交差点のヒートマップ（選択されている場合）の交点数の線形および半対数プロットが生成されます（ 図10 ）。あまり発達していない腺は、同じMEA...

Watch this Scientific Journal Video about Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method at JoVE.com

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

げっ歯類における乳腺発達は、典型的には、記述的評価または基本的物理的属性の測定によって評価されている。分枝密度は、客観的に定量することが困難な哺乳類の発達の指標である。このプロトコールは、乳腺分枝特性の定量的評価のための信頼できる方法を記載している。

ショール分析法を用いたラット乳腺全身の分枝密度の定量

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

quantifying-branching-density-rat-mammary-gland-whole-mounts-using

Research

JoVE Journal

Developmental Biology

14.2K ビュー.  National Institute of Environmental Health Sciences. げっ歯類における乳腺発達は、典型的には、記述的評価または基本的物理的属性の測定によって評価されている。分枝密度は、客観的に定量することが困難な哺乳類の発達の指標である。このプロトコールは、乳腺分枝特性の定量的評価のための信頼できる方法を記載している。 

ビデオ: Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

エバンスブルー色素でゼブラフィッシュ幼生骨格筋の整合性の分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

発生生物学

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

リンパ腺や血球中を調べるための方法<em&gt;ショウジョウバエ</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

で活躍ERKの時空間解析<em&gt; C。エレガンス</em&gt;生殖細胞系列

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

軟骨細胞分化のDNAメチル化レベルを定量化する5-mCドットブロットアッセイ<em&gt;インビトロ</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

細胞外小胞/エキソソームを介した乳腺上皮細胞と乳房管腔細胞との間の乳腺形成能の伝達

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

マウス乳腺のギャップ、タイトおよびアデュランス接合部の成分間のタンパク質 - タンパク質相互作用および共局在の分析

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

血管内皮幹細胞の血管形成を視覚化するための新規乳腺脂肪移植技術

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

ノードと走査型電子顕微鏡と蛍光抗体法マウント全体を使用して Gastrulating のマウス胚における脊索板を可視化

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

緑藻クラミドモナス・ラインハルトティの同期培養物の詳細分析のためのマルチオミクス抽出法

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...

ショール分析法を用いたラット乳腺全身の分枝密度の定量

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