Name: sectioning mammary gland whole mounts for lesion identification
Uploaded: 2017-07-24T14:00:00
Description: Watch this Scientific Journal Video about Sectioning Mammary Gland Whole Mounts for Lesion Identification Video at JoVE.com

The authors would like to acknowledge Pam Ovwigho, Tenette Jones, and Natasha Clayton, NIEHS, for their technical expertise and support with this project.

All animal use and procedures for this study were approved by the NIEHS Laboratory Animal
Care and Use Committee and conducted in an Association for Assessment and Accreditation of
Laboratory Animal Care-accredited facility.
1. Mammary Whole-mount Preparation2,3,6,7
<ol>
	<li>Remove the inguinal mammary glands from one side of a non-pregnant CD-1 female mouse, as describe...

<ol>
	<li>Rudel, R. A., Fenton, S. E., Ackerman, J. M., Euling, S. Y., Makris, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+exposures+and+mammary+gland+development:+State+of+the+science,+public+health,+implications,+and+research+recommendations.">Environmental exposures and mammary gland development: State of the science, public health, implications, and research recommendations.</a> Environ Health Perspect. 119 (8), 1053-1061 (2011).</li><li>Keenan, K. P., Wallig, M. A., Hascheck, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+via+Nurture:+Effect+of+Diet+on+Health,+Obesity,+and+Safety+Assessment.">Nature via Nurture: Effect of Diet on Health, Obesity, and Safety Assessment.</a> Toxicol Pathol. 41 (2), 190-209 (2013).</li><li>Stanko, J. P., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+branching+density+in+rat+mammary+gland+whole+mounts+using+the+Sholl+analysis+method.">Quantifying branching density in rat mammary gland whole mounts using the Sholl analysis method.</a> J Vis Exp. , (2017).</li><li>Filgo, A. J., et al. <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+evaluation+in+juvenile+toxicity+studies:+Temporal+developmental+patterns+in+the+male+and+female+Harlan+Sprague+Dawley+Rat.">Mammary gland evaluation in juvenile toxicity studies: Temporal developmental patterns in the male and female Harlan Sprague Dawley Rat.</a> Toxicol Pathol. 44 (7), 1034-1058 (2016).</li><li>Tucker, D. K., Foley, J. K., Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+high-quality+hematoxylin+and+eosin-stained+sections+from+rodent+mammary+gland+whole+mounts+for+histopathologic+review.">Preparation of high-quality hematoxylin and eosin-stained sections from rodent mammary gland whole mounts for histopathologic review.</a> Toxicol Pathol. 44 (7), 1059-1064 (2016).</li><li>Hvid, H., Thorup, O., Oleksiewicz, M. B., Sjogren, I., Jensen, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+preparation+of+tissue+sections+from+the+rat+mammary+gland.">An alternative method for preparation of tissue sections from the rat mammary gland.</a> Exp Toxicol Path. 63 (4), 317-324 (2011).</li><li>Munoz-de-Toro, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinatal+exposure+to+bisphenol-A+alters+peripubertal+mammary+gland+development+in+mice.">Perinatal exposure to bisphenol-A alters peripubertal mammary gland development in mice.</a> Endocrinology. 146 (9), 4138-4147 (2005).</li><li>Padilla-Banks, E., Jefferson, W. N., Newbold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+exposure+to+the+phytoestrogen+genistein+alters+mammary+gland+growth+and+developmental+programming+of+hormone+receptor+levels.">Neonatal exposure to the phytoestrogen genistein alters mammary gland growth and developmental programming of hormone receptor levels.</a> Endocrinology. 147 (10), 4871-4882 (2006).</li><li>Nikaido, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+maternal+xenotestrogen+exposure+on+development+of+the+reproductive+tract+and+mammary+gland+in+female+CD-1+mouse+offspring.">Effects of maternal xenotestrogen exposure on development of the reproductive tract and mammary gland in female CD-1 mouse offspring.</a> Reprod Toxicol. 18 (6), 803-811 (2004).</li></ol>

The mammary gland whole mount is a powerful tool that may be used to illustrate the normal mammary development and morphological alterations that may arise and persist following exposure to chemicals, including endocrine disruptors. When a whole mount and H&#38;E section from the same animal are assessed together, they can provide an accurate visual snapshot of early alterations, which can progress into a more diseased state.
The ability to obtain this useful information lies in ensuring that ...

The mammary whole mountis a useful and inexpensive method implemented in many rat and mouse studies to understand both normal and chemically-induced, abnormal development. In general, a mammary gland collected at various stages during rodent development (i.e., adolescence, puberty, mid- to late-gestation, and the involution phase) will show morphological changes in tissue and cellular architecture influenced by paracrine, endocrine, and autocrine factors1. In the aging rat, the epithelial and stromal portions of the gland become increasingly dense, which makes measuring morphological parameters difficult in a whole mount. Thus, it is common practice to collect a gland for histological evaluation to identify changes on a microscopic level. Both processes are especially useful in studies involving chemical exposure (i.e., environmental, or pharmaceutical) or to examine the morphological changes accompanied by genetic alterations.
Techniques and capabilities for using the mammary gland in risk evaluation continue to evolve. While whole-mount preparation is routine and standardized, modifications to sectioning continue2,3. Many research groups from various backgrounds (i.e., academia, government, and industry) have adapted coronal/longitudinal sections of mammary tissue as a preferred method, while some labs still use cross-cut sections through the skin4. The latter method results in an inordinate representation of epidermis (skin), rather than the tissue of interest: the mammary gland epithelium2. Coronal or longitudinal sections are more useful for characterizing normal tissue5 and greatly improve the detection of abnormalities and lesions due to the increased surface area and number of structures present. Overall, this makes the whole mount a suitable method for the comparative histopathological assessment of the mammary gland1,2. Whether using a mouse or a rat, retrieval, and preservation of the 4th and 5th inguinal glands is highly recommended for the comparison of the whole mount to a contralateral H&#38;E section.
When used in combination, the H&#38;E section and whole mount provide an accurate account of the cellular and morphological changes induced by environmental exposure. This is especially true in certain rodent strains in which the model possesses a low susceptibility to tumor formation or a tumor is not grossly visible. In some circumstances, obtaining the required tissue may not always be possible using both techniques (i.e., insufficient tissue quantity, resources, or unexpected experimental results). In our case, abnormalities were observed in the mammary whole mount, whereas histopathological findings in the contralateral H&#38;E gland were mostly normal. The overwhelming discrepancy between the contralateral glands led us to develop an economical and efficient procedure to identify the abnormalities in the whole mounts. Using a modified processing and embedding procedure, high-quality H&#38;E-stained sections were prepared from paraffin-embedded whole mounts. We envision this protocol becoming a powerful detection tool for chemical exposure effects, enhancing inter-lab comparisons.

<table border="0" cellpadding="0" cellspacing="0" width="1013">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Xylene</td>
			<td style="width:309px;">Sigma-Aldrich</td>
			<td style="width:152px;">214736</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Permount</td>
			<td>Thermo Fisher Scientific</td>
			<td>SP15-100</td>
			<td>mounting media for coverslipping whole mounts and H&#38;E sections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Leica automated processor</td>
			<td>Leica Biosystems</td>
			<td>Model # ST5020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">HM 355S Automatic Microtome</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>905200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Paraffin</td>
			<td>Leica Biosystems</td>
			<td style="width:152px;">EM-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Superfrost plus microscope slides</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:152px;">4951PLUS-001</td>
			<td>electrostatically charged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Fisher Finest 24x60x1</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:152px;">12-548-5P</td>
			<td>coverslips</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Varistain Gemini ES Automatic slide stainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>A78000014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Sta-On</td>
			<td>Leica Biosystems</td>
			<td>3803107</td>
			<td>liquid adhesive; used in water bath at sectioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modified Harris Hematoxylin 72711</td>
			<td>Richard-Allan Scientific (Part of Thermo Scientific)</td>
			<td>72711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Eosin</td>
			<td>Leica Biosystems</td>
			<td>3801600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Lithium Carbonate</td>
			<td>SkyTech Laboratories</td>
			<td style="width:152px;">LCQ500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Glacial Acetic Acid</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:152px;">A38-500</td>
			<td>needed in Carnoy&#39;s fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Chloroform</td>
			<td>Macron Fine Chemicals</td>
			<td style="width:152px;">4440-04</td>
			<td>needed in Carnoy&#39;s fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">100% Ethanol</td>
			<td>The Warner Graham Company</td>
			<td style="width:152px;">6505001050000</td>
			<td>needed in Carnoy&#39;s fixative and washing slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">95% Ethanol</td>
			<td>The Warner Graham Company</td>
			<td style="width:152px;">6505011137320190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Carmine Alum</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">C1022-25G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:385px;">Aluminum potassium sulfate dodecahydrate, Sigma Ultra</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">A7210-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Automation wash buffer, 20X</td>
			<td>Biocare Medical</td>
			<td>TWB945</td>
			<td></td>
		</tr>
	</tbody>
</table>

sectioning mammary gland whole mounts for lesion identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

This method is effective at assisting with diagnoses that may have otherwise been missed if the original contralateral H&#38;E section of the mammary pad did not show any histological changes. However, the outcome will only be useful if care is taken during the initial whole-mount preparation, as well as during the preparation of the tissue for the histological evaluation. Paraffin embedding will provide protection and will help to preserve the tissue for future sectioning.
<p class="...

Watch this Scientific Journal Video about Sectioning Mammary Gland Whole Mounts for Lesion Identification Video at JoVE.com

Sectioning Mammary Gland Whole Mounts for Lesion Identification Video (Video) | JoVE

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Research

JoVE Journal

Cancer Research

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis Video (Video) | JoVE

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization Video (Video) | JoVE

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas Video (Video) | JoVE

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad Video (Video) | JoVE

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Extraction of Extracellular Vesicles from Whole Tissue

Extraction of Extracellular Vesicles from Whole Tissue Video (Video) | JoVE

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland Video (Video) | JoVE

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad Video (Video) | JoVE

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells Video (Video) | JoVE

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research Video (Video) | JoVE

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis Video (Video) | JoVE

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...