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

1. Die Entfernung der Abdominal Brustdrüse und Vorbereiten einer Whole Berg <ol><li> Euthanize das Tier nach dem IACUC Richtlinien. Pin das Tier auf seinem Rücken aus den Beinen mit Nadeln oder Klebeband die Beine auf die Oberfläche Bord, und wischen Sie die Haut nass mit Ethanol (EtOH). Heben Sie die Haut ein wenig mit einer Pinzette und mit einer scharfen Schere einen Medianschnitt in die Haut, die zwischen dem Paar Nr. 5 Brustwarzen und schneiden gegen den Hals. Machen Sie ein umgekehrtes Y Einschnitt von der Mittellinie in Richtung der Hinterbeine. Wenn Thorax Milchdrüsen auch gesammelt werden sollen, machen auch ein Y Einschnitt von der Mittellinie zwischen den beiden # 2 Nippel auf die Vorderbeine. </li><li> Präparieren Sie die Haut mit einer Schere auf einer Seite offen der invertierten Y Schnitt mit der Bauchwand Nr. 4 Brustdrüse aussetzen, die Milchdrüsen sind auf der Haut angebracht. Pin der Haut mit Nadeln auf der Oberfläche Bord, vollständig freizulegen der Drüse. Die Arbeit der einen Seite und Drüse an der Zeit. </li><li> Präparieren Sie die Brustdrüse frei von der Haut entweder mit einer scharfen Schere und / oder ein Skalpell, ausgehend von der proximalen Bereich in der Nähe der Brustwarze und Arbeiten in Richtung des distalen Endes der Drüse in Richtung der Wirbelsäule des Tieres. (Dies kann einige Minuten dauern, vor allem in einer älteren Ratte.) </li><li> Sofort verbreitete das freistehende Drüse auf eine entsprechend beschriftete Objektträger aus Glas (mit einem richtigen Filzstift Stift und / oder &quot;ein Diamant pen&quot;), und die Ausbreitung der Drüse sorgfältig entsprechend seiner ursprünglichen Größe und Form in situ. Die Glasplatte muss größer sein als die Drüse. Nach Ausbreitung der Drüse auf die Folie, lassen Sie es auf den Tisch zu setzen für eine Weile, so dass die Drüse auf der Folie klebt, aber nicht trocknen lassen. </li><li> Legen Sie die Folie in ein Gefäß mit Carnoy Fixativ (75% Eisessig, 25% absolutem Ethanol = EtOH), lassen Sie es bei Raumtemperatur (RT) in den Abzug, der für 2 Tage oder länger fest zu bekommen. Dias können auch in das Fixiermittel für einen längeren Zeitraum belassen werden. </li><li> Waschen Sie die Slides in 70% EtOH für 1 Stunde bei RT. </li><li> Spülen in destilliertem Wasser für 30 min bei RT. </li><li> Stain in Carmine Alum Fleck *) für 2 Tage oder länger (bis Sie sehen, dass die Lymphknoten durch gefärbt; Blick auf die Rückseite der Folie). </li><li> Wash in zunehmenden Reihe von EtOH, 1 Stunde in jeder: 70% -&gt; 95% -&gt; 100%. Nach der letzten Waschung in absolutem EtOH stellen die Drüsen in Xylol. Lassen Sie sitzen in den Dunstabzug in RT mindestens für 2 Tage. Dieser letzte Schritt ist der Brustdrüse Bedeutung Delipidierung der Brustfettpolster Lichtung, und die anschließende Erhöhung der Transparenz. Die fetthaltiger der Drüse ist die längere Clearing Zeit benötigt wird. </li><li> Berg mit Deckgläschen mit einer Montageplatte Medien, wie Permount. Lassen Sie die Folien gut trocknen (mehrere Tage), bevor die Beobachtung unter einem Stereomikroskop. * Carmine Alum Stain: Platz 1 g Karmin (Sigma C1022) und 2,5 g Aluminium-Kalium-Sulfat (Sigma A7167) in 500 ml destilliertem Wasser und kochen für 20 min. Passen Endvolumen von 500 mL mit Wasser. Filter und kalt stellen. Die Lösung kann für mehrere Monate eingesetzt werden. Verwerfen, wenn Farbe schwach wird. </li></ol> 2. Analyse der Brustdrüse Whole Mount Morphologie Brustdrüse ganzen mounts Morphologie ist nach der folgenden Endpunkte und die Ergebnisse mit Brustkrebs Risiko korreliert analysiert. <ol><li> Brustdrüse ganzen Berg an PND 21 (präpubertären Alter): <ul><li> Epitheliale Wachstum: <ul><li> Entfernung von der Brustwarze bis zum Ende des epithelialen Baum (in Millimetern), gemessen mit einem Lineal </li></ul></li><li> Potenzial für eine maligne Transformation: <ul><li> Anzahl der TEBS (terminalen Ende Knospen), zählte unter einem Lichtmikroskop. TEBS sind die größten bauchige Strukturen nur am distalen Ende des Brustepithelzellen Baum entfernt </li></ul></li><li> Differenzierung: <ul><li> AB1 (alveoläre bud) Partitur (0-5). Alveolar Knospen sind über das Epithel ausbreiten. </li><li> AB2 Score (0-5) </li></ul></li></ul> Ganze montiert sind unter einem Lichtmikroskop gezählt. Die Score-Werte von AB1 und AB2 sind für eine endgültige Differenzierung addiert. </li><li> Brustdrüse ganzen Berg an PND 50 (post-pubertären Alter) gesammelt: <ul><li> Epitheliale Wachstum: <ul><li> Entfernung der Lymphknoten bis zum Ende des epithelialen Baum (in Millimetern), gemessen mit einem Lineal. </li><li> Entfernung von der Spitze der epithelialen Baum bis zum Ende der Fettpolster, gemessen mit einem Lineal. </li></ul></li><li> Potenzial für eine maligne Transformation: <ul><li> Anzahl der TEBS, unter einem Lichtmikroskop gezählt </li></ul></li><li> Differenzierung: <ul><li> AB1 Score (0-5) </li><li> AB2 Score (0-5) </li><li> Läppchen Score (0-5) </li></ul></li></ul> Ganze montiert sind unter einem Lichtmikroskop gezählt. Differenzierung wird in zweierlei Hinsicht beurteilt. Erstens sind die Score-Werte von AB1, AB2 und Läppchen für eine endgültige Differenzierung addiert. Darüber hinaus wird das Verhältnis zwischen Läppchen Partitur und AB1 + AB2 Score berechnet werden. ABs zu Läppchen zu unterscheiden, und je höher das Verhältnis zwischen Läppchen und ABS, je differenzierter die Drüse. </li></ol> 3. Palpation und Brusttumor Measurement in Ratten und Korrelation zum Milchdrüse Morphologie <ol><li> Zu Beginn dieses Verfahren, halten die Ratte durch Ergreifen der ganze Körper mit der Handfläche über den Rücken, mit dem Zeigefinger hinter dem Kopf und dem Daumen und dem zweiten Finger unter das Gegenteil Achselhöhle. Schalten Sie die Ratte so dass es an seiner hinteren lügt, und tasten jede Brusttumoren zu entdecken; tastbaren Tumoren sollte wie ein &quot;Klumpen&quot; zu fühlen. </li><li> Next mit einem Messschieber die Breite, Länge und Höhe des Tumors zu messen. Berechnen Sie das Volumen des Tumors mit dem Ellipsoid Formel, Volume = 1/6πabc, wo &#39;a&#39; = Breite, &#39;b&#39; = Länge und &#39;c&#39; = Höhe. </li><li> Manuelles Eintragen der Lage und Größe des Tumors in einem Notebook auf einer wöchentlichen Basis. Diese Daten werden in eine Tabelle übertragen später. </li></ol> 4. Repräsentative Ergebnisse Eine sorgfältige Präparation des Brustfettpolster und ihre Verarbeitung zu wholemount wird die Abschätzung des Entwicklungsstandes der Brustdrüse. Wenn jeder Schritt richtig ausgeführt wird, ist die ganze Brustepithelzellen Baum deutlich sichtbar in der Fettpolster, und dies ermöglicht die einfache Bestimmung der Anzahl der TEBS und Berechnung der Dichte des alveolären Knospen und Läppchen. Fehltritte bei der Vorbereitung wholemounts können Fehler, die ganze Fettpolster, ungenügende Befestigung und unzureichende Clearing sezieren. Beurteilung der Brustdrüse Morphologie gibt Auskunft über die Anzahl der vorhandenen Strukturen, die zu Mammatumoren (TEBS), Grad der Differenzierung der epithelialen Strukturen (alveoläre Knospen und Läppchen), und messen des Wachstums (duktales Dehnung) geben kann. Es ist wichtig, TEBS zur terminalen Enden zu trennen; die letzteren sind am distalen Ende des Epithels befindet sich, ähnlich wie bei TEBS, aber sie sind kleiner als TEBS und keinen Anlass zu bösartigen Tumoren. Terminalen Enden sind auch innerhalb der epithelialen Baum gesehen. <img src="/files/ftp_upload/2260/2260fig1.jpg" alt="Abbildung 1" /> Abbildung 1:. Repräsentatives Ergebnis von gut vorbereitet Brustdrüse whole mount Alle Komponenten der Drüse wurden ordnungsgemäß zerlegt und Carmin-Färbung ist optimal. <img src="/files/ftp_upload/2260/2260fig2.jpg" alt="Abbildung 2" /> Abbildung 2:. Repräsentatives Ergebnis einer schlecht vorbereiteten Brustdrüse whole mount Die Drüse wurde ordnungsgemäß zerlegt (den distalen Teil der Drüse fehlt), wurde nicht richtig gedehnt und Delipidierung noch nicht vollständig eingetreten ist.

<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>

Beurteilung der Brustdrüse morphologischen Endpunkten und das Wachstum der epithelialen Baum kann verwendet werden, um vorherzusagen, ob eine frühe Leben diätetische Manipulationen oder sonstige Manipulationen, die in utero oder präpubertären hormonelle Umfeld verändern, modifizieren später Brustkrebs Risiko. Da Brustkrebs bei Menschen in Brust-Strukturen (terminale duktale lobuläre Einheiten, TDLUs) ähnlich TEBS bei Ratten ausgelöst wird, kann diese Technik verwendet, um das Potenzial des frühen Lebens Forderungen an das Brustkrebsrisiko beeinflussen zu bestimmen. Weitere, in den Frauen hohe mammographische Dichte erhöht das Risiko für Brustkrebs um 4-6 fache, und Beurteilung des Wachstums der Brustepithelzellen Baum kann verwendet werden, um Faktoren, die mammographische Dichte und Faktoren, die diese Dichte zu reduzieren bestimmen zu identifizieren. Um diese Informationen zu erhalten, sollte Brustdrüse voll zerlegt werden, ordnungsgemäß auf einem Glasträger, so dass alle seine Komponenten können unter dem Mikroskop sichtbar gestreckt. Darüber hinaus werden geeignete carmine Färbung und Delipidierung in Xylol liefern ganzen mounts, die sich für Brustkrebs Risikobewertung sind.

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<td>Glacial acetic acid</td>
<td>&nbsp;</td>
<td>EM</td>
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<td>Absolute ethanol</td>
<td>&nbsp;</td>
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<td>Carmine</td>
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<td>Aluminum potassium sulfate</td>
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changes in mammary gland morphology and breast cancer risk in rats

Studien in Tiermodellen von Brustkrebs zeigen, dass die Forderungen der Nahrung / hormonelle Faktoren während der in utero und pubertären Zeiten, in denen der Brustdrüse umfangreiche Modellierungs-und Re-Modellierung erfährt, verändern die Anfälligkeit für karzinogen-induzierten Mammatumoren. Ähnliche Ergebnisse wurden beim Menschen beschrieben worden: zum Beispiel erhöht hohen Geburtsgewicht später Risiko an Brustkrebs zu erkranken, und der Aufnahme von Soja in der Kindheit verringert Brustkrebs-Risiko. Es wird vermutet, dass diese prä-und postnatalen Änderungen der Ernährung anhaltenden morphologischen Veränderungen in der Brustdrüse, die wiederum zu ändern Brustkrebsrisiko im späteren Leben zu induzieren. Diese morphologischen Veränderungen wahrscheinlich reflektieren epigenetische Modifikationen, wie zum Beispiel Änderungen in der DNA-Methylierung, Histon-und miRNA-Expression, die dann Auswirkungen auf Gen-Transkription. In diesem Artikel beschreiben wir, wie sich Änderungen in Brustdrüse Morphologie Brustkrebs Risiko bei Ratten vorherzusagen. Unser Protokoll ausdrücklich beschreibt, wie zu sezieren und entfernen Sie die Ratte abdominale Brustdrüse und wie Brustdrüse ganzen mounts vorzubereiten. Es beschreibt auch, wie Brustdrüse Morphologie nach drei Endpunkte (Anzahl der terminalen Ende Knospen-, Epithel-Dehnung und Differenzierung) und die Daten verwenden, um Risiko der Entwicklung von Brustkrebs vorhersagen zu analysieren.

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

Unser Protokoll beschreibt, wie die Ratte abdominale Brustdrüse sezieren und wie Brustdrüse ganzen mounts vorzubereiten. Es beschreibt auch, wie Brustdrüse Morphologie mit drei Endpunkten (Anzahl der terminalen Ende Knospen-, Epithel-Dehnung und Differenzierung) zu analysieren und diese Ergebnisse nutzen, um Brustkrebs Risiko bei Ratten, die zu Änderungen der Ernährung ausgesetzt waren vorhersagen<em&gt; In utero</em&gt; Oder während Vorpubertät.

Änderungen der Brustdrüsen Morphologie und Breast Cancer Risk in Ratten

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 Aufrufe.  Georgetown University. Unser Protokoll beschreibt, wie die Ratte abdominale Brustdrüse sezieren und wie Brustdrüse ganzen mounts vorzubereiten. Es beschreibt auch, wie Brustdrüse Morphologie mit drei Endpunkten (Anzahl der terminalen Ende Knospen-, Epithel-Dehnung und Differenzierung) zu analysieren und diese Ergebnisse nutzen, um Brustkrebs Risiko bei Ratten, die zu Änderungen der Ernährung ausgesetzt waren vorhersagen In utero Oder während Vorpubertät.

Serie: 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.

Evaluation der Brustdrüse Entwicklung und Funktion in Mausmodellen

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 ...

Forschung

Biologie

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.

Quantitative Messung der Invadopodia-vermittelte extrazellulären Matrix Proteolyse in Einzel-und Multizelluläre Contexts

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.

Ein analytisches Werkzeug, das zelluläre Morphologie Änderungen von Dreidimensionale Fluorescence Images Quantifiziert

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).

Kombiniert Immunfluoreszenz und DNA FISH auf 3D erhaltene Interphase Nuclei, um Änderungen in 3D Nuclear Organization Study

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.

Intraduktale Injection für Lokalisierte Drug Delivery auf die Maus Milchdrüse

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.

Die indirekte Immunfluoreszenz auf Gefrierschnitte von Mäusemilchdrüse

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.

Vergleich von drei verschiedenen Methoden zur Bestimmung Zellproliferation in Brustkrebs-Zelllinien

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.

Isolierung von intakt, ganze Maus Milchdrüsen zur Analyse der extrazellulären Matrix Ausdruck und Drüse Morphologie

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.

Bovine Brustdrüse Biopsie-Techniken

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.

Längsschnittliche intravitale Mikroskopie unter Verwendung eines Brustbildgebungsfensters mit austauschbarem Deckel

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