NCI (U54 CA00100970), NCI (1 R03 CA150040-01), ACS (116 602-PF-09-018-01-CNE)

1. Ablation de la glande mammaire et abdominale Préparation d&#39;un mont entier <ol><li> Euthanasier l&#39;animal selon les directives du IACUC. Pin de l&#39;animal sur le dos de ses jambes avec des aiguilles, ou du ruban adhésif les jambes sur le bord de surface, et essuyer la peau mouillée avec de l&#39;éthanol (EtOH). Soulever la peau un peu avec des pinces et de ciseaux pointus faire une incision médiane dans la peau de départ entre la paire n ° 5 mamelons et couper vers le cou. Faire une incision Y inversé de la ligne médiane vers les pattes postérieures. Si les glandes mammaires thoraciques doivent également être recueillis, font aussi une incision Y de la ligne médiane entre la paire n ° 2 mamelons vers les pattes avant. </li><li> Disséquer la peau avec des ciseaux ouverts sur un côté de l&#39;incision Y inversé pour exposer la glande abdominale # 4 mammaire; les glandes mammaires sont attachés à la peau. Pin de la peau avec des aiguilles sur le bord de surface, complètement exposer la glande. Le travail d&#39;un côté et la glande à l&#39;époque. </li><li> Disséquer la glande mammaire gratuite à partir de la peau soit en utilisant des ciseaux pointus et / ou d&#39;un scalpel, à partir de la zone proximale proche de l&#39;embout et le travail vers l&#39;extrémité distale de la glande vers la colonne vertébrale de l&#39;animal. (Cela peut prendre plusieurs minutes, surtout dans un vieux rat.) </li><li> Immédiatement répandre la glande détachée sur une lame de verre convenablement étiquetés (utilisation d&#39;un marqueur approprié permanente et / ou &#39;un stylo diamant »), et la propagation de la glande attentivement correspondant à sa taille d&#39;origine et la forme in situ. La lame de verre doit être plus grand que la glande. Après la diffusion de la glande sur la lame, laissez-le reposer sur la table pour un moment, alors que la glande bâtons sur la lame, mais ne laissez pas sécher. </li><li> Mettez le glisser dans un bocal contenant fixateur de Carnoy (75% d&#39;acide acétique glacial, 25% d&#39;éthanol absolu = EtOH), laissez-vous fixé à température ambiante (TA) dans la hotte pour 2 jours ou plus. Les diapositives peuvent aussi être laissé dans le fixateur pour une plus longue période de temps. </li><li> Laver les lames dans EtOH à 70% pendant 1 heure à température ambiante. </li><li> Rincer à l&#39;eau distillée pendant 30 min à température ambiante. </li><li> Colorer dans une tache de carmin d&#39;alun *) pour 2 jours ou plus (jusqu&#39;à ce que vous voyez que les ganglions lymphatiques ont taché grâce, regardez à l&#39;arrière de la lame). </li><li> Laver en série croissante d&#39;EtOH, 1 heure dans chaque: 70% -&gt; 95% -&gt; 100%. Après le dernier lavage dans EtOH absolu mettre les glandes dans le xylène. Laissez reposer dans la hotte en RT au moins pour 2 jours. Cette dernière étape est la compensation de la délipidation sens glande mammaire de la plaquette mammaires graisse, et augmentation subséquente de la transparence. La plus grasse de la glande est le plus long temps de compensation est nécessaire. </li><li> Monter avec des lamelles en utilisant un média de montage, comme Permount. Laissez bien sécher les diapositives (plusieurs jours) avant d&#39;observer sous un microscope stéréo. * Alun Carmine taches: Placer 1 g de carmin (Sigma C1022) et 2,5 g de sulfate de potassium en aluminium (Sigma A7167) dans 500 ml d&#39;eau distillée et faire bouillir pendant 20 min. Ajuster le volume final à 500 ml avec de l&#39;eau. Filtre et réfrigérer. Solution peut être utilisée pendant plusieurs mois. Jetez quand la couleur devient faible. </li></ol> 2. L&#39;analyse de la morphologie mammaire Gland mont entier Mammaire toute la glande monte morphologie est analysée selon les critères suivants end-points et les résultats en corrélation avec le risque de cancer mammaire. <ol><li> Glande mammaire toute monture au PND 21 (pré-pubères âge): <ul><li> De croissance épithélial: <ul><li> Distance de la tétine à la fin de l&#39;arbre épithéliales (en millimètres), mesurée à l&#39;aide d&#39;une règle </li></ul></li><li> Potentiel de transformation maligne: <ul><li> Nombre de TEBS (bourgeons extrémité terminale), comptés sous un microscope optique. TEBS sont les plus grandes structures bulbeuses situé à seulement à l&#39;extrémité distale de l&#39;arbre épithéliales mammaires </li></ul></li><li> Différenciation: <ul><li> AB1 (alvéolaire bourgeon) score (0-5). Bourgeons alvéolaires sont réparties à travers l&#39;épithélium. </li><li> AB2 score de (0-5) </li></ul></li></ul> Monte Entières sont marqués d&#39;un microscope optique. Les valeurs de score d&#39;AB1 et AB2 sont ajoutés pour un score de différenciation finale. </li><li> Glande mammaire toute monture collectées au PND 50 (post-pubères âge): <ul><li> De croissance épithélial: <ul><li> Distance à partir du noeud lymphatique à la fin de l&#39;arbre épithéliales (en millimètres), mesurée à l&#39;aide d&#39;une règle. </li><li> Distance de la pointe de l&#39;arbre de l&#39;épithélium à la fin du coussinet adipeux, mesurée à l&#39;aide d&#39;une règle. </li></ul></li><li> Potentiel de transformation maligne: <ul><li> Nombre de TEBS, comptés sous un microscope optique </li></ul></li><li> Différenciation: <ul><li> AB1 score de (0-5) </li><li> AB2 score de (0-5) </li><li> Score lobules (0-5) </li></ul></li></ul> Monte Entières sont marqués d&#39;un microscope optique. La différenciation sera évalue de deux manières. Tout d&#39;abord, les valeurs score de AB1, AB2 et lobules sont ajoutés pour un score de différenciation finale. En outre, le ratio entre le score lobules et AB1 AB2 + score sera calculé. ABs différencier pour lobules, et plus le rapport entre les lobules et ABS, le plus différencié de la glande est. </li></ol> 3. La palpation et la mesure de tumeur mammaire chez le rat et la corrélation de la morphologie glande mammaire <ol><li> Pour commencer cette procédure, maintenez le rat en saisissant l&#39;ensemble du corps avec la paume sur le dos, avec l&#39;index derrière la tête et le pouce et le majeur sous l&#39;aisselle opposée. Tournez le rat il est couché sur son postérieur, et palper pour détecter toute tumeurs mammaires, tumeurs palpables devrait se sentir comme un «forfaitaire». </li><li> Suivant l&#39;utilisation d&#39;un pied à mesurer la largeur, la longueur et la hauteur de la tumeur. Calculez le volume de la tumeur en utilisant la formule ellipsoïde, Volume = 1/6πabc, où &#39;a&#39; = largeur, la longueur &#39;b&#39; =, et &#39;c&#39; = hauteur. </li><li> Enregistrer manuellement l&#39;emplacement et la taille de la tumeur dans un carnet sur une base hebdomadaire. Ces données seront transférées vers un tableur tard. </li></ol> 4. Les résultats représentatifs Une dissection minutieuse du pad mammaires graisse et son traitement au wholemount permettra d&#39;évaluer l&#39;état de développement de la glande mammaire. Lorsque chaque étape est effectuée correctement, l&#39;ensemble des arbres épithéliales mammaires est clairement visible dans le coussinet adipeux, ce qui permet de déterminer facilement le nombre de TEBS et le calcul de la densité des bourgeons alvéolaires et lobules. Faux pas dans les échantillons entiers préparation peut inclure l&#39;échec de disséquer le pavé toute la graisse, la fixation insuffisante, et la compensation inadéquate. Évaluation de la morphologie de la glande mammaire va fournir des informations sur le nombre de présents des structures qui peuvent donner lieu à des tumeurs mammaires (TEBS), le degré de différenciation des structures épithéliales (bourgeons alvéolaires et lobules), et mesure de la croissance (élongation canalaire). Il est important de séparer TEBS aux extrémités terminales; ces derniers sont situés à l&#39;extrémité distale de l&#39;épithélium, à l&#39;instar TEBS, mais ils sont plus petits que TEBS et ne donnent pas naissance à des tumeurs malignes. Extrémités terminales sont également vus dans l&#39;arbre épithéliales. <img src="/files/ftp_upload/2260/2260fig1.jpg" alt="Figure 1" /> Figure 1:. Résultat représentatif du bien préparés monter la glande mammaire de toute Tous les composants de la glande ont été correctement disséqués et coloration carmin est optimale. <img src="/files/ftp_upload/2260/2260fig2.jpg" alt="Figure 2" /> Figure 2:. Résultat représentatif d&#39;une monture mal préparés glande mammaire toute la glande a été correctement disséquées (la partie distale de la glande est manquante), n&#39;a pas été correctement tendue et délipidation n&#39;a pas complètement eu lieu.

<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>

Évaluation de la glande mammaire morphologique points d&#39;extrémité et la croissance de l&#39;arbre épithéliales peut être utilisé pour prédire si des manipulations diététiques début de la vie, ou d&#39;autres manipulations qui modifient l&#39;environnement in utero ou prépubères hormonaux, de modifier le risque de cancer plus tard mammaire. Puisque le cancer du sein chez l&#39;homme est initié dans les structures mammaires (unités terminales lobulaire canalaire, TDLUs) similaire à TEBS chez le rat, cette technique peut être utilisée pour déterminer le potentiel de l&#39;exposition précoce à affecter le risque de cancer du sein. En outre, chez les femmes de haute densité mammographique augmente le risque de cancer du sein par 4-6 fois, et l&#39;évaluation de la croissance des arbres épithéliales mammaires peuvent être utilisés pour identifier les facteurs qui déterminent la densité mammographique et des facteurs qui réduisent cette densité. Pour obtenir cette information, la glande mammaire devrait être entièrement disséqué, bien tendue sur une lame de verre afin que tous ses composants peuvent être visualisées sous un microscope. En outre, bon coloration carmin et délipidation dans le xylène fournira monte entier qui sont adaptés à l&#39;évaluation du risque de cancer mammaire.

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<td>Glacial acetic acid</td>
<td>&nbsp;</td>
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<td>Absolute ethanol</td>
<td>&nbsp;</td>
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<td>Carmine</td>
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<td>Sigma</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

Des études dans des modèles rongeurs d&#39;un cancer du sein montrent que l&#39;exposition à des facteurs alimentaires / hormonaux pendant la période in utero et pubertaire, lorsque la glande mammaire subit la modélisation vaste et re-modélisation, de modifier la sensibilité aux carcinogènes induits des tumeurs mammaires. Des constatations similaires ont été décrits chez l&#39;homme: par exemple, poids élevé augmente le risque ultérieur de développer un cancer du sein, et l&#39;apport alimentaire de soja durant l&#39;enfance diminue le risque de cancer du sein. On pense que ces modifications prénatals et postnatals alimentaires induisent des changements morphologiques persistant dans la glande mammaire qui à leur tour modifier le risque de cancer du sein plus tard dans la vie. Ces changements morphologiques reflètent probablement des modifications épigénétiques, comme des changements dans la méthylation de l&#39;ADN, les histones et l&#39;expression du miARN qui affectent alors la transcription des gènes. Dans cet article nous décrivons comment les changements dans la morphologie des glandes mammaires peut prédire le risque de cancer mammaire chez les rats. Notre protocole décrit précisément comment disséquer et enlever la glande abdominale du rat mammaire et la façon de préparer monte glande mammaire ensemble. Il décrit également la façon d&#39;analyser la morphologie des glandes mammaires en fonction de trois points d&#39;extrémité (nombre de bourgeons extrémité terminale, l&#39;élongation et la différenciation épithéliale) et d&#39;utiliser les données pour prédire le risque de développer un cancer mammaire.

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

Notre protocole décrit comment disséquer le rat des glandes mammaires et abdominales comment préparer monte glande mammaire ensemble. Il décrit également la façon d&#39;analyser la morphologie des glandes mammaires à partir de trois points d&#39;extrémité (nombre de bourgeons extrémité terminale, l&#39;élongation et la différenciation épithéliale) et d&#39;utiliser ces résultats pour prédire le risque de cancer mammaire chez les rats qui ont été exposés à des modifications diététiques<em&gt; In utero</em&gt; Ou pendant prépuberté.

Changements dans la morphologie glande mammaire et le risque de cancer du sein chez les rats

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 ...

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29.4K vues.  Georgetown University. Notre protocole décrit comment disséquer le rat des glandes mammaires et abdominales comment préparer monte glande mammaire ensemble. Il décrit également la façon d'analyser la morphologie des glandes mammaires à partir de trois points d'extrémité (nombre de bourgeons extrémité terminale, l'élongation et la différenciation épithéliale) et d'utiliser ces résultats pour prédire le risque de cancer mammaire chez les rats qui ont été exposé...

Vidéo: 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 de développement des glandes mammaires et fonction dans des modèles murins

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 ...

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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.

Mesure quantitative de invadopodes la protéolyse matrice extracellulaire dans des contextes simples et multicellulaire

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.

Un outil analytique qui quantifie les changements de morphologie cellulaire en trois dimensions des images de fluorescence

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).

Immunofluorescence combinés et FISH ADN sur 3D préservé noyaux en interphase pour étudier les changements dans l&#39;organisation nucléaire 3D

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.

Injection intra-canalaire pour localisée Drug Delivery de la glande mammaire de souris

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.

Immunofluorescence indirecte sur des coupes congelées de glande mammaire de souris

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.

Comparaison de trois différentes méthodes pour la détermination de la prolifération cellulaire dans des lignées cellulaires de cancer du sein

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.

Isolement des glandes mammaires de souris intacte, toute analyse d’Expression de la matrice extracellulaire et la morphologie de la glande

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.

Techniques de biopsie des glandes mammaires bovine

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.

Microscopie intravitale longitudinale à l’aide d’une fenêtre d’imagerie mammaire avec couvercle remplaçable

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