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.
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
2. Removal of the Mammary Whole Mount from a Glass Slide
3. Tissue Processing
4. Embedding the Processed Mammary Tissue
5. Sectioning the Paraffin-embedded Mammary Tissue on the Microtome
6. Automated and Manual H&E Staining of Sectioned Mammary Tissues
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.
To determine the ideal thickness of the mammary tissue, 4-µm and 6-µm sections were prepared. We tested depths that were greater than the margin of error of ± 1 µm on the microtome. Sections of a 6-µm thickness (Figure 1A) were very dense with compact cells . Overall, the slides lacked distinct cellular details that would be necessary to make a proper identification. The optimal thickness was 4 µm (Figure 1B). These tissues provided the best results, where epithelial and stromal areas and corresponding cell types were easily distinguished.
Slides from a large, ongoing study were used to illustrate the ease and utility of this method. In several cases, the original H&E section from 14-month-old virgin female CD-1 offspring were found to have conflicting findings compared to the contralateral mammary whole mount from the same animal. Lesions were evident in the whole mount, but could not be identified without further sectioning and staining. Samples from two different animals were chosen as representative cases. In both cases, a single section was used for staining, but several cuts were made until a representative section was obtained. Very few cuts were necessary to obtain a representative section, since previous whole-mount preparations cleared most of the fat pad, leaving the gland very thin compared to a mammary gland originally prepared for paraffin embedding, which is surrounded by a thick fat pad. Figure 2A and Figure 3A illustrate histological sections of glands that were assessed as normal. Both glands show ductal structures surrounded by a robust and homogenous adipocyte-rich population. Each duct is lined by a single layer of simple cuboidal epithelial cells and is maintained by a second layer of basal cells, mainly composed of myoepithelial cells, but also encompassing stem and progenitor cell populations. The representative contralateral whole mounts (Figures 2B and Figure 3B) demonstrated ducts and stroma with increased opacity. However, it was difficult to determine if the opaqueness was the result of hyperplastic, inflammatory, or neoplastic alterations without having an H&E section that could provide distinct cellular details.
Contralateral whole mounts from the same animals were used to implement the method described here and can be observed in Figure 2C and D and Figure 3C and D. The samples in Figure 2C & D were diagnosed as perivascular inflammation due to the increased number of lymphocytes that were present around a large blood vessel in the mammary gland section. For the second case, the mammary gland lobular architecture was maintained but was enlarged multifocally by the increased number and size of normal alveoli and ducts (lobuloalveolar hyperplasia). Alveolar epithelial cells were well-differentiated, round, often-vacuolated, and formed one concentric layer around a lumen that typically contained proteinaceous fluid. Ducts were lined by columnar cells and were similar to alveolar cells, with a well-differentiated epithelium that formed one concentric layer. This phenotype is most often observed in the mammary glands of mid-pregnant mice and rats. This should not be confused with lobuloalveolar structures in the mammary glands of adult male rats8. In the adult male mammary gland, alveoli are prominent and ducts are infrequent, but the alveoli and ducts are lined by stratified epithelium, with tall, vacuolated cuboidal to short columnar epithelial cells.
Figure 1:Recommended thickness for mouse mammary gland whole mount to H&E sections. A) 6 µm and B) 4 µm. The resolutions of mammary gland structures were not optimal for histopathological evaluation using the thicker (6-µm) sections, so the 4-µm section is recommended. This figure has been modified from Tucker et al.9. Please click here to view a larger version of this figure.
Figure 2: Whole mount to H&E section of perivascular inflammation. This image is a mouse mammary gland that was collected in diestrus. A) Formalin-fixed H&E mammary gland section with no histopathogical alterations. B) Contralateral whole-mount section, with areas of increased opacity (boxed area). C) 20x magnification of an H&E section from a mammary whole mount (boxed area). Clusters of mononuclear cells surrounded the blood vessel and extended into the surrounding adipose tissue. D) The 40x magnification of an H&E section from a mammary whole mount (boxed area) shows in greater detail that most of the mononuclear population consists of lymphocytes and highlights the severity of the perivascular inflammation. This figure has been modified from Tucker et al.9. Please click here to view a larger version of this figure.
Figure 3: Whole mount to H&E section of lobular alveolar hyperplasia. This image is a mouse mammary gland that was collected in diestrus. A) Formalin-fixed H&E mammary gland section with no histopathological alterations. B) Contralateral whole-mount section with increased opacity in the ductal and stromal areas (boxed area). C) 20x magnification of an H&E section from a mammary whole mount (boxed area). Lobular architecture was maintained but was enlarged multifocally by the increased number and size of normal alveoli and ducts (lobuloalveolar hyperplasia). D) The 40x magnification of an H&E section from a mammary whole mount (boxed area) reveals that the enlarged lobules contain an increased number of alveoli and ducts that are lined by well-differentiated, often-vacuolated epithelial cells, which form lumens that contain proteinaceous fluid. This figure has been modified from Tucker et al.9. Please click here to view a larger version of this figure.
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 care is taken to preserve and not compromise glandular morphology when removing the tissue. While the rodent has several mammary gland sites located bilaterally along the dorsal wall, it is recommended to collect the 4th and 5th glands to minimize muscle tissue recovery. The nipple attachment area and lymph node should also be present, as they serve as useful morphological landmarks. The gland should also be spread and stretched across a flat surface (i.e., a glass slide, cardstock, or cloth) to closely mimic the natural structure of the tissue in situ. Abnormalities within the mammary gland are usually not visible until after the gland has been defatted in xylene or has been carmine-stained. Unlike mammary tissue that was originally prepared for histological sectioning, a whole mount removed from the glass for paraffin embedding will be extremely thin and fragile. Forceps impressions and tissue tears should be avoided, as they will possibly alter cellular morphology and make histopathological evaluations of the finished product difficult.
Once the whole mount has been paraffin-embedded, prior to sectioning, it is recommended to allow the blocks to incubate at -20 °C for 1 h. This step improves the ability to obtain an optimal representative tissue section in a ribbon. Sectioning of the tissue at 6 µm (Figure 1A)9 produced a dense cellular architecture, with less-than-optimal resolution. By using a thinner section (Figure 1B)9, all cellular features, including epithelial tissue and surrounding stromal infiltrates, were easily identified. It should also be noted that, although these sections were previously carmine-stained, the stain was not visible following sectioning and did not interfere with H&E staining. Therefore, a de-staining step was not necessary in the protocol. Although tissue sections were stained solely with H&E, we expect with minor modifications, other histochemical and possibly immunohistochemical stains might be applicable once sectioning is complete. However, it was beyond the extent of this protocol and will require further investigation to determine the optimal conditions for these stains.
This procedure was developed because we observed discrepant findings between routinely collected whole mounts and contralateral H&E-stained mammary gland sections from the same animal. Similar mammary protocols exist6,10; however, the procedures were not detailed or easy to follow. Those methods established a basis for the development of our protocol. Normal gland morphology was observed in the H&E-stained tissue sections (Figure 2A and Figure 3A)1, while the complementary whole mounts revealed numerous abnormal morphological features (Figure 2B and Figure 3B)9. By performing histology on the whole mounts, we could classify the abnormal features. For example, perivascular inflammation and lobuloalveolar hyperplasia were identified in two separate cases that were disparate compared to the H&E findings observed in the contralateral sides (Figure 2C and D and Figure 3C and D)9.
To the authors' knowledge, there are no known reports of mammary gland discrepancies similar to those observed in our ongoing study. However, this may be due to experimental design issues rather than lack of occurrence, as the mammary gland is often solely collected for one application or analysis that does not involve morphology, such as RT-PCR or Western blots. However, many experiments incorporate multiple applications that require an adequate amount of tissue for each endpoint. The inguinal glands are the preferred choice for whole mounts and downstream applications because they seldom have contaminating surrounding tissue like the thoracic glands (i.e., muscle contamination) and because the inguinal mammary lymph nodes provide valuable landmarks for orientation and comparison across glands. Therefore, deciding which gland and how much of the gland will be dedicated to each application will influence the precision of detection. Prioritization for the use of the mammary gland should be for: 1) a whole mount composed of the entire 4th and 5th glands, 2) histology of the contralateral 4th gland containing some lymph node for use as a landmark, and 3) the contralateral 5th gland (with no contaminating lymph node) for downstream applications (i.e., RNA, DNA, and protein). If an abnormality is visible at necropsy, a whole mount should take preference for histology collection.
As with any protocol, there are accompanying limitations; the need to permanently destroy the whole mount during sectioning and having limited tissue for use in alternative analyses, for example. Thus, we recommend extensively documenting whole mounts with a desktop scanner to produce digital images for future analysis and reference. Also, if staining will not occur within a month, limit the number of sections cut to preserve the tissue for any future analyses. The benefits of this whole mount to H&E section method outweigh the limitations, such that it can be universally applied to mammary studies involving multiple disciplines, especially toxicology. Several studies using chemicals with known endocrine effects have included a modified version of this protocol, demonstrating its applicability11,12,13. Findings from these studies may help to reduce the chances of a false negative in chemical testing but may also provide new or additional information that can be used for regulatory and risk-assessment decisions.
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 |