Instituto Nacional del Cáncer (U54 CA00100970), Instituto Nacional del Cáncer (1 R03 CA150040-01), ACS (116602-PF-09-018-01-CNE)

1. Extirpación de la glándula mamaria abdominal y Preparación de un monte se <ol><li> La eutanasia de los animales de acuerdo con las directrices IACUC. Pin del animal en la espalda de sus piernas con las agujas, o la cinta de las piernas sobre el tablero de la superficie, y limpie la piel húmeda con etanol (EtOH). Levante la piel un poco con pinzas y con unas tijeras afiladas hacer una incisión en la piel partida entre los dos pezones # 5 y cortar hacia el cuello. Hacer una incisión en Y invertida de la línea media hacia las patas traseras. Si torácica glándulas mamarias son también para ser recogidos, hacer también una incisión Y de la línea media entre el par # 2 pezones hacia las patas delanteras. </li><li> Disección de la piel con una tijera abierta en un lado de la incisión en Y invertida para exponer la glándula mamaria abdominal # 4; las glándulas mamarias se adhiere a la piel. Pin de la piel con agujas en el tablero de la superficie, para exponer completamente la glándula. El trabajo de un lado y de la glándula en el momento. </li><li> Disecar la glándula mamaria, libre de la piel, ya sea usando unas tijeras afiladas y / o un bisturí, a partir de la zona proximal cerca del pezón y de trabajo hacia el extremo distal de la glándula hacia la espina dorsal del animal. (Esto puede tardar varios minutos, especialmente en una rata más.) </li><li> Inmediatamente se extendió la glándula separado en un portaobjetos de vidrio debidamente etiquetados (usar un lápiz adecuado marcador permanente y / o &quot;una pluma de diamantes&quot;), y la propagación de la glándula cuidado correspondiente a su tamaño y forma original in situ. La lámina de vidrio debe ser mayor que la glándula. Después de la difusión de la glándula en la diapositiva, dejar que repose en la mesa por un rato, para que la glándula se pega en la diapositiva, pero no deje que se seque. </li><li> Poner la diapositiva en un frasco que contiene el fijador de Carnoy (75% de ácido acético glacial, 25% de etanol absoluto = EtOH), dejar que se fija a la temperatura ambiente (TA) en la campana extractora de humos de 2 días o más. Diapositivas también se pueden dejar en el fijador durante un largo periodo de tiempo. </li><li> Se lavan los portas en un 70% EtOH durante 1 hora a temperatura ambiente. </li><li> Enjuague con agua destilada durante 30 min a temperatura ambiente. </li><li> Mancha en Alum Carmine mancha *) durante 2 días o más (hasta que vea que los ganglios linfáticos se han manchado a través, buscar en la parte posterior de la diapositiva). </li><li> De lavado en serie creciente de EtOH, 1 hora en cada uno: 70% -&gt; 95% -&gt; 100%. Después del último lavado en EtOH absoluto puesto que las glándulas en xileno. Deje reposar en la campana extractora de humos en RT por lo menos durante 2 días. Este último paso es la limpieza de la delipidación glándula mamaria significado de la almohadilla de grasa mamaria, y el consiguiente aumento de la transparencia. El más grasa de la glándula es el mayor tiempo de limpieza es necesaria. </li><li> Montar con cubreobjetos utilizando un medio de montaje, como Permount. Deje secar bien las diapositivas (varios días) antes de observar bajo un microscopio estereoscópico. * Alum Carmine manchas: Coloque 1 g de carmín (Sigma C1022) y 2,5 g de aluminio sulfato de potasio (Sigma A7167) en 500 ml de agua destilada y hervir durante 20 minutos. Ajustar el volumen final de 500 ml con agua. Filtro y el refrigerador. Solución puede ser utilizada por varios meses. Desechar cuando el color se debilita. </li></ol> 2. Análisis de la morfología de la glándula mamaria monte entero Toda la glándula mamaria se monta la morfología se analiza de acuerdo a los siguientes criterios de valoración y los resultados correlacionados con el riesgo de cáncer de mama. <ol><li> Glándula mamaria todo el montaje en el PND 21 (pre-púberes edad): <ul><li> Crecimiento epitelial: <ul><li> Distancia desde el pezón hasta el final del árbol epiteliales (en milímetros), medido con una regla </li></ul></li><li> Potencial de transformación maligna: <ul><li> Número de TEBS (yemas terminales final), se contaron bajo un microscopio de luz. TEBS son las mayores estructuras de bulbo situado a tan sólo en el extremo distal del árbol epiteliales mamarias </li></ul></li><li> Diferenciación: <ul><li> AB1 (alveolar yema) puntuación (0-5). Brotes alveolares se propagan a través del epitelio. </li><li> AB2 puntuación (0-5) </li></ul></li></ul> Montajes conjuntos se anotó con un microscopio óptico. El valor de la puntuación de AB1 y AB2 se añaden a una puntuación final de la diferenciación. </li><li> Glándula mamaria todo el montaje recogidos en PND 50 (post-puberal edad): <ul><li> Crecimiento epitelial: <ul><li> Distancia desde el ganglio linfático hasta el final del árbol epiteliales (en milímetros), medido con una regla. </li><li> Distancia desde la punta del árbol del epitelio hasta el final de la almohadilla de grasa, medido con una regla. </li></ul></li><li> Potencial de transformación maligna: <ul><li> Número de TEBS, contados bajo un microscopio de luz </li></ul></li><li> Diferenciación: <ul><li> AB1 puntuación (0-5) </li><li> AB2 puntuación (0-5) </li><li> Lóbulos puntuación (0-5) </li></ul></li></ul> Montajes conjuntos se anotó con un microscopio óptico. La diferenciación se evalúa de dos maneras. En primer lugar, las puntuaciones de AB1, AB2 y lobulillos se añaden a una puntuación final de la diferenciación. Además, la relación entre la puntuación de los lobulillos y los resultados AB1 + AB2 será calculada. ABs diferenciar a los lóbulos, y la mayor es la relación entre los lobulillos y los abdominales, el más diferenciado de la glándula. </li></ol> 3. Palpación y valoración del tumor de mama en ratas y la correlación con morfología de la glándula mamaria <ol><li> Para iniciar este procedimiento, mantenga la rata, sujetando el cuerpo con la palma de la mano sobre la espalda, con el dedo índice detrás de la cabeza y el pulgar y el segundo bajo la axila opuesta. A su vez la rata por lo que está mintiendo en su posterior, y palpar para detectar tumores de mama, los tumores palpables debe sentirse como un &quot;bulto&quot;. </li><li> Luego, use una pinza para medir la anchura, longitud y altura del tumor. Calcular el volumen del tumor utilizando la fórmula del elipsoide de volumen, = 1/6πabc, donde &#39;a&#39; = anchura, la longitud &#39;b&#39; = y &#39;c&#39; = altura. </li><li> Manual de registro de la ubicación y el tamaño del tumor en un cuaderno sobre una base semanal. Estos datos serán transferidos a una hoja de cálculo más tarde. </li></ol> 4. Resultados representante Disección cuidadosa de la almohadilla de grasa mamaria y su procesamiento para wholemount permitirá evaluar el estado de desarrollo de la glándula mamaria. Cuando cada paso se realiza correctamente, el conjunto de árboles epiteliales mamarias es claramente visible en la almohadilla de grasa, lo que permite determinar fácilmente el número de TEBS y el cálculo de la densidad de brotes alveolares y lobulillos. Errores en la preparación de wholemounts pueden incluir falta de analizar toda la almohadilla de grasa, la fijación insuficiente, y la limpieza inadecuada. Evaluación de la morfología de la glándula mamaria proporcionará información sobre el número de las actuales estructuras que pueden dar lugar a tumores de mama (TEBS), grado de diferenciación de las estructuras epiteliales (brotes alveolares y lóbulos), y la medida del crecimiento (alargamiento ductal). Es importante separar TEBS a los fines de terminales, este último se encuentra en el extremo distal del epitelio, de manera similar a TEBS, pero son más pequeños que TEBS y no dan lugar a tumores malignos. Extremos terminales también se ven en el árbol del epitelio. <img src="/files/ftp_upload/2260/2260fig1.jpg" alt="Figura 1" /> Figura 1:. Representante de resultado bien preparado montar toda la glándula mamaria Todos los componentes de la glándula han sido debidamente disecados y manchas de carmín es óptima. <img src="/files/ftp_upload/2260/2260fig2.jpg" alt="Figura 2" /> Figura 2:. Representante resultado de un mal preparados montar toda la glándula mamaria La glándula ha sido debidamente disecada (la porción distal de la glándula no se encuentra), no se ha estirado bien y delipidación no se ha producido por completo.

<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>

Evaluación de la glándula mamaria morfológicas puntos finales y el crecimiento del árbol epiteliales se puede utilizar para predecir si las manipulaciones tempranas de la vida la dieta, u otras manipulaciones que alteran el medio ambiente en el útero o prepuberal hormonal, modificar el riesgo de cáncer de mama más tarde. Dado que el cáncer de mama en humanos se inicia en las estructuras mamarias (unidades de terminal ductal lobular, TDLUs), similar a TEBS en ratas, esta técnica se puede utilizar para determinar el potencial de las exposiciones de la primera infancia para afectar el riesgo de cáncer de mama. Además, en las mujeres la densidad mamográfica de alta aumenta el riesgo de cáncer de mama por 4-6 veces, y la evaluación del crecimiento de árboles epiteliales mamarias se puede utilizar para identificar los factores que determinan la densidad mamográfica y los factores que reducen la densidad. Para obtener esta información, la glándula mamaria debe estar completamente disecados, bien tirante en un portaobjetos de vidrio de modo que todos sus componentes pueden ser visualizados con un microscopio. Además, adecuada coloración carmín y delipidación en xileno proporcionará toda monta que son adecuados para la evaluación del riesgo de cáncer de mama.

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<td>Glacial acetic acid</td>
<td>&nbsp;</td>
<td>EM</td>
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<td>Carnoy's Fixative</td>
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<td>Absolute ethanol</td>
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<td>Carmine</td>
<td>&nbsp;</td>
<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

Los estudios en modelos de roedores de cáncer de mama muestran que la exposición a factores de la dieta / hormonal durante el período en el útero y la pubertad, cuando la glándula mamaria sufre de modelado amplia y re-modelado, alterar la susceptibilidad a carcinógenos inducida por tumores mamarios. Hallazgos similares han sido descritos en los seres humanos: por ejemplo, peso al nacer aumenta más alto riesgo de desarrollar cáncer de mama y la ingesta de soja durante la infancia reduce el riesgo de cáncer de mama. Se cree que estas modificaciones en la dieta prenatal y postnatal persistente inducir cambios morfológicos en la glándula mamaria que, a su vez modificar el riesgo de cáncer de mama en el futuro. Estos cambios morfológicos probablemente se deben a modificaciones epigenéticas, como los cambios en la metilación del ADN, histonas y la expresión de miRNA que luego afectan la transcripción de genes. En este artículo se describe cómo los cambios en la morfología de la glándula mamaria puede predecir el riesgo de cáncer de mama en ratas. El protocolo se describe específicamente cómo analizar y eliminar las ratas de la glándula mamaria abdominal y la forma de preparar montajes de la glándula mamaria completa. También se describe la forma de analizar la morfología de la glándula mamaria de acuerdo con tres puntos finales (número de yemas terminales de final, la elongación y la diferenciación del epitelio) y utilizar los datos para predecir el riesgo de desarrollar cáncer mamario.

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

El protocolo describe cómo diseccionar la rata de las glándulas mamarias abdominales y la forma de preparar montajes de la glándula mamaria completa. También se describe la forma de analizar la morfología de la glándula mamaria con tres puntos finales (número de yemas terminales de final, la elongación y la diferenciación del epitelio) y utilizar estos resultados para predecir el riesgo de cáncer de mama en ratas que fueron expuestas a modificaciones en la dieta<em&gt; En el útero</em&gt; O durante la prepubertad.

Los cambios en la morfología de la glándula mamaria y el riesgo de cáncer de mama en ratas

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

29.4K vistas.  Georgetown University. El protocolo describe cómo diseccionar la rata de las glándulas mamarias abdominales y la forma de preparar montajes de la glándula mamaria completa. También se describe la forma de analizar la morfología de la glándula mamaria con tres puntos finales (número de yemas terminales de final, la elongación y la diferenciación del epitelio) y utilizar estos resultados para predecir el riesgo de cáncer de mama en ratas que fueron expuestas a modificaciones en la dieta...

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.

Evaluación de la glándula mamaria de desarrollo y función en modelos de ratón

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 ...

Investigación

Biología

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.

La medición cuantitativa de invadopodios mediada por proteolisis de la matriz extracelular en contextos simples y multicelulares

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.

Una herramienta de análisis que cuantifica los cambios celulares Morfología de imágenes tridimensionales de fluorescencia

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).

Inmunofluorescencia y FISH Combinado de ADN en 3D conservados núcleos en interfase para estudiar los cambios en la organización nuclear 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.

Inyección intraductal de entrega de la droga localizada a la glándula mamaria de ratón

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.

Inmunofluorescencia indirecta en secciones congeladas de ratón Glándula Mamaria

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.

Comparación de tres diferentes métodos para determinar la proliferación celular en líneas celulares de cáncer de mama

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.

Aislamiento de las glándulas mamarias del ratón intacto, todo para el análisis de expresión de la matriz extracelular y la morfología de la glándula

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.

Técnicas de biopsia de glándula mamaria 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.

Microscopía intravital longitudinal con una ventana de imágenes mamarias con tapa reemplazable

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