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.
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.
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&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.
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 …
The authors have nothing to disclose.
The authors would like to acknowledge Pam Ovwigho, Tenette Jones, and Natasha Clayton, NIEHS, for their technical expertise and support with this project.
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 |