Sectioning Mammary Gland Whole Mounts for Lesion Identification

Summary

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.

Abstract

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&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&E section.  The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&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&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&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.

Introduction

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 continue^2,3. 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 gland^1,2. Whether using a mouse or a rat, retrieval, and preservation of the 4^th and 5^th inguinal glands is highly recommended for the comparison of the whole mount to a contralateral H&E section.

When used in combination, the H&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&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&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.

Protocol

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 Remove the inguinal mammary glands from one side of a non-pregnant CD-1 female mouse, as describe…

Representative Results

This method is effective at assisting with diagnoses that may have otherwise been missed if the original contralateral H&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="…

Discussion

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&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 …

Materials

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