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

1. La rimozione della ghiandola mammaria e addominale Preparazione di un monte intero <ol><li> Eutanasia degli animali secondo le linee guida IACUC. Perno l&#39;animale al suo ritorno da gambe con aghi o nastro le gambe sul bordo della superficie, e pulire la pelle umida con etanolo (EtOH). Sollevare la pelle un po &#39;con una pinza e con forbici affilate fare una incisione mediana nella pelle di partenza tra la coppia # 5 capezzoli e tagliare verso il collo. Fare un&#39;incisione a Y rovesciata dalla linea mediana verso le zampe posteriori. Se toracica ghiandole mammarie sono da raccogliere, fare anche un&#39;incisione Y dalla linea mediana tra la coppia # 2 capezzoli verso le zampe anteriori. </li><li> Sezionare la pelle aperto con le forbici su un lato l&#39;incisione a Y rovesciata per esporre la ghiandola mammaria addominale # 4, le ghiandole mammarie sono attaccati alla pelle. Perno la pelle con aghi sulla tavola di superficie, per esporre completamente la ghiandola. Lavoro da un lato e la ghiandola al momento. </li><li> Sezionare la ghiandola mammaria libero dalla pelle usando forbici affilate e / o un bisturi, a partire dalla zona prossimale vicino al capezzolo e di lavoro verso la fine distale della ghiandola verso la colonna vertebrale dell&#39;animale. (Questo può richiedere alcuni minuti, specialmente in un ratto vecchio.) </li><li> Immediatamente diffondere la ghiandola staccata su un vetrino adeguatamente etichettati (con una penna adeguata pennarello indelebile e / o &#39;una penna di diamante&#39;), e diffondere la ghiandola attentamente corrispondente alla sua dimensione originale e la forma in situ. Il vetrino deve essere più grande della ghiandola. Dopo la diffusione della ghiandola sul vetrino, lasciare riposare sul tavolo per un po &#39;, in modo che la ghiandola bastoni sul vetrino, ma non lasciate asciugare. </li><li> Mettere il vetrino in un vaso contenente fissativo di Carnoy (75% di acido acetico glaciale, 25% di etanolo assoluto = EtOH), lasciare che si fissa a temperatura ambiente (RT) nella cappa per 2 giorni o più. Diapositive può anche essere lasciato in fissativo per un periodo di tempo più lungo. </li><li> Lavare i vetrini in 70% EtOH per 1 ora a temperatura ambiente. </li><li> Lavare in acqua distillata per 30 minuti a RT. </li><li> Colorare con Alum Carmine macchia *) per 2 giorni o più (fino a vedere che i linfonodi hanno macchiato attraverso; guardare il retro della diapositiva). </li><li> Lavare in serie crescente di EtOH, 1 ora ciascuno: 70% -&gt; 95% -&gt; 100%. Dopo l&#39;ultimo lavaggio in assoluto EtOH mettere le ghiandole in xilene. Lasciate riposare nella cappa in RT almeno per 2 giorni. Questo ultimo passo è di compensazione del delipidation ghiandola mammaria significato del cuscinetto di grasso mammaria, e conseguente aumento della trasparenza. La più grassa la ghiandola è il più lungo tempo di decolorazione è richiesto. </li><li> Montare con cover-scivola con un altro supporto di montaggio, come Permount. Lasciate asciugare bene le diapositive (alcuni giorni) prima di osservare al microscopio stereo. * Allume Carmine Stain: Mettere 1 g di carminio (Sigma C1022) e 2,5 g di potassio solfato di alluminio (Sigma A7167) in 500 ml di acqua distillata e fate bollire per 20 min. Regolare il volume finale a 500 ml con acqua. Filtrare e conservare in frigorifero. Soluzione può essere utilizzata per diversi mesi. Scartare quando il colore diventa debole. </li></ol> 2. Analisi di Monte ghiandola mammaria Morfologia Whole Tutta la ghiandola mammaria monta morfologia viene analizzata secondo i seguenti end-point ed i risultati correlati con il rischio di cancro mammario. <ol><li> Ghiandola mammaria intera-mount a PND 21 (pre-puberale età): <ul><li> Crescita epiteliale: <ul><li> Distanza dal capezzolo al fine di albero epiteliale (in millimetri), misurata con un righello </li></ul></li><li> Potenziale di trasformazione maligna: <ul><li> Numero di TEBS (gemme fine terminale), contate al microscopio ottico. TEBS sono le più grandi strutture bulbose trova solo al termine distale della struttura epiteliali mammarie </li></ul></li><li> Differenziazione: <ul><li> AB1 (alveolare gemma) punteggio (0-5). Gemme alveolare sono diffuse in tutta l&#39;epitelio. </li><li> AB2 punteggio (0-5) </li></ul></li></ul> Monti interi sono segnati con un microscopio ottico. I valori di punteggio da AB1 e AB2 sono aggiunti per un punteggio finale di differenziazione. </li><li> Ghiandola mammaria intera-mount raccolti in PND 50 (post-puberale età): <ul><li> Crescita epiteliale: <ul><li> Distanza dal linfonodo alla fine di albero epiteliale (in millimetri), misurata con un righello. </li><li> Distanza dalla punta dell&#39;albero epiteliale alla fine del cuscinetto adiposo, misurata con un righello. </li></ul></li><li> Potenziale di trasformazione maligna: <ul><li> Numero di TEBS, contate al microscopio ottico </li></ul></li><li> Differenziazione: <ul><li> AB1 punteggio (0-5) </li><li> AB2 punteggio (0-5) </li><li> Lobuli punteggio (0-5) </li></ul></li></ul> Monti interi sono segnati con un microscopio ottico. La differenziazione sarà valuta in due modi. In primo luogo, i valori di punteggio da AB1, AB2 e lobuli sono aggiunti per un punteggio finale di differenziazione. Inoltre, il rapporto tra il punteggio lobuli e AB1 + AB2 punteggio verrà calcolato. AB differenziare a lobuli, e maggiore è il rapporto tra lobuli e AB, il più differenziato è la ghiandola. </li></ol> 3. Palpazione e misurazione tumore mammario nei ratti e Correlazione di Morfologia ghiandola mammaria <ol><li> Per iniziare questa procedura, tenere il ratto afferrando tutto il corpo con il palmo sul dorso, con l&#39;indice dietro la testa e il pollice e secondo dito sotto l&#39;ascella opposta. Girare il ratto quindi è sdraiato sul suo posteriore, e palpare di rilevare eventuali tumori mammari, tumori palpabili dovrebbe sentire come un &quot;grumo&quot;. </li><li> Avanti utilizzare un calibro per misurare la larghezza, la lunghezza e l&#39;altezza del tumore. Calcolare il volume del tumore utilizzando la formula dell&#39;ellissoide, Volume = 1/6πabc, dove &#39;a&#39; = larghezza, lunghezza = &#39;b&#39;, e &#39;c&#39; = altezza. </li><li> Registrare manualmente la posizione e le dimensioni del tumore in un quaderno su base settimanale. Questi dati saranno trasferiti a un foglio di calcolo più tardi. </li></ol> 4. Rappresentante Risultati Dissezione attenta del cuscinetto di grasso mammaria e la sua elaborazione per wholemount permetterà accertamento del suo stato di sviluppo della ghiandola mammaria. Quando ogni passo è fatto correttamente, l&#39;intero albero epiteliali mammarie è chiaramente visibile all&#39;interno del cuscinetto adiposo, e questo permette una facile determinazione del numero di TEBS e calcolo della densità di gemme alveolare e lobuli. Passi falsi in wholemounts preparazione possono includere il mancato sezionare il pad tutto grasso, fissaggio insufficiente, inadeguata e di compensazione. Valutazione della morfologia della ghiandola mammaria fornirà informazioni sul numero di presenti strutture che possono dare origine a tumori mammari (TEBS), grado di differenziazione delle strutture epiteliali (gemme alveolare e lobuli), e la misura della crescita (allungamento duttale). È importante separare TEBS a fini terminale, la seconda si trova alla fine distale dell&#39;epitelio, analogamente a TEBS, ma sono più piccoli TEBS e non danno luogo a tumori maligni. Le estremità terminali sono visto anche all&#39;interno della struttura epiteliale. <img src="/files/ftp_upload/2260/2260fig1.jpg" alt="Figura 1" /> Figura 1:. Rappresentante del risultato ben preparati montare tutto ghiandola mammaria Tutti i componenti della ghiandola sono stati adeguatamente sezionato e colorazione carminio è ottimale. <img src="/files/ftp_upload/2260/2260fig2.jpg" alt="Figura 2" /> Figura 2:. Rappresentante risultato di un monte mal preparato tutto ghiandola mammaria La ghiandola è stato correttamente sezionato (la porzione distale della ghiandola è mancante), non è stato adeguatamente allungata e delipidation non è completamente avvenuta.

<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>

Nessun conflitto di interessi dichiarati.

Valutazione della ghiandola mammaria morfologiche end-point e la crescita dell&#39;albero epiteliale può essere usato per prevedere se manipolazioni primi anni di vita alimentari, o altre manipolazioni che alterano in ambiente utero o in età prepuberale ormonali, modificare il rischio di cancro mammario in seguito. Dato che il cancro al seno negli esseri umani è iniziato in strutture mammaria (unità terminali duttale lobulare, TDLUs) simile a TEBS nei ratti, questa tecnica può essere usata per determinare il potenziale di esposizioni primi anni di vita di influenzare il rischio di cancro al seno. Inoltre, nelle donne ad alta densità mammografica aumenta il rischio di cancro al seno di 4-6 volte, e la valutazione della crescita di alberi epiteliali mammarie possono essere utilizzate per identificare i fattori che determinano la densità mammografica e fattori che riducono la densità. Per ottenere queste informazioni, ghiandola mammaria dovrebbe essere completamente sezionato, opportunamente allungato su un vetrino in modo che tutti i suoi componenti possono essere visualizzati al microscopio. Inoltre, proprio colorazione carminio e delipidation in xilene fornirà supporti intero che sono adatti per la valutazione del rischio di cancro mammario.

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

Studi in modelli di roditori di spettacolo cancro al seno che l&#39;esposizione a fattori dietetici / ormonali durante il periodo in utero e puberale, quando la ghiandola mammaria subisce modellazione estesa e ri-modellazione, alterare la suscettibilità ai tumori mammari cancerogeno indotto. Risultati simili sono stati descritti negli esseri umani: per esempio, alto peso alla nascita aumenta poi il rischio di sviluppare il cancro al seno, e assunzione di soia durante l&#39;infanzia si riduce il rischio di cancro al seno. Si pensa che queste modifiche prenatali e postnatali alimentari inducono persistenti cambiamenti morfologici nella ghiandola mammaria, che a loro volta modificano il rischio di cancro al seno nel corso della vita. Questi cambiamenti morfologici probabilmente riflettono modifiche epigenetiche, quali i cambiamenti nella metilazione del DNA, istoni e l&#39;espressione miRNA che poi influenzano la trascrizione del gene. In questo articolo viene descritto come i cambiamenti nella morfologia della ghiandola mammaria può predire il rischio di cancro mammario nei ratti. Il nostro protocollo descrive specificatamente come sezionare e rimuovere la ghiandola mammaria di ratto addominale e come preparare i supporti ghiandola mammaria intero. Descrive anche come analizzare la morfologia della ghiandola mammaria in base a tre end-point (numero di gemme fine terminale, allungamento epiteliali e la differenziazione) e di utilizzare i dati per predire il rischio di sviluppare il cancro mammario.

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

Il nostro protocollo descrive come sezionare la ghiandola mammaria di ratto addominale e come preparare monta ghiandola mammaria intero. Descrive anche come analizzare la morfologia della ghiandola mammaria utilizzando tre end-point (numero di gemme fine terminale, allungamento epiteliali e la differenziazione) e di utilizzare questi risultati per predire il rischio di cancro mammario nei ratti che sono stati esposti a modificazioni dietetiche<em&gt; In utero</em&gt; O durante prepubertà.

Cambiamenti nella morfologia della ghiandola mammaria e rischio di cancro al seno in topi

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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JoVE Journal

Biology

29.4K Views.  Georgetown University. Il nostro protocollo descrive come sezionare la ghiandola mammaria di ratto addominale e come preparare monta ghiandola mammaria intero. Descrive anche come analizzare la morfologia della ghiandola mammaria utilizzando tre end-point (numero di gemme fine terminale, allungamento epiteliali e la differenziazione) e di utilizzare questi risultati per predire il rischio di cancro mammario nei ratti che sono stati esposti a modificazioni dietetiche In utero O du...

Video: 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.

Valutazione dello Sviluppo ghiandola mammaria e funzione in modelli murini

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 ...

Ricerca

Biologia

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.

Misurazione quantitativa della Invadopodia-mediata proteolisi della matrice extracellulare in singoli contesti e pluricellulari

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.

Uno strumento di analisi che quantifica Modifiche morfologia cellulare dalle immagini a fluorescenza tridimensionali

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).

Immunofluorescenza combinato e FISH DNA 3D conservato nuclei in interfase di studiare i cambiamenti in 3D Organizzazione nucleare

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.

Iniezione intraduttale per Localizzato Drug Delivery per il mouse ghiandola mammaria

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.

Immunofluorescenza indiretta su sezioni congelate di mouse ghiandola mammaria

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.

Confronto di tre diversi metodi per la determinazione proliferazione cellulare in Breast Cancer linee cellulari

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.

Isolamento delle ghiandole mammarie Mouse intatto, intero per l'analisi di espressione di matrice extracellulare e la morfologia della ghiandola

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.

Tecniche di biopsia della ghiandola mammaria bovina

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.

Microscopia intravitale longitudinale utilizzando una finestra di imaging mammario con coperchio sostituibile

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