Rats were maintained in an AAALAC-approved facility on a 12 h light/dark cycle and provided LabDiet 5001 Rodent Diet (PMI® Nutrition International) and water ad libitum. Inbred Wistar Furth (WF/Hsd) females were obtained from Envigo. All animal protocols were approved by the University of Louisville Animal Care and Use Committee.
DMBA Administration and Mammary Gland Tissue Resection
At 50-55 days of age 7,12-Dimethylbenz(a)anthracene (DMBA Acros Organics, CAS#: 57-97-6, 20 mg/mL in sesame oil) was given by single oral gavage (65 mg DMBA/kg body mass) to WF/Hsd (Envigo) females to induce mammary carcinogenesis. Mammary glands were resected four or twelve weeks following DMBA administration. Briefly, one skin incision was made at the lower abdomen and extended forward, along the midline, to the neck. Four additional skin incisions were made, each extending from the midline down each limb. Skin was separated from the body cavity to reveal mammary glands present in pairs along each ventro-lateral boundary of the body. The larger mammary glands, such as the combined thoracic pectoral glands toward the anterior (Fig 1, labeled B/C glands) or individual abdominal-inguinal glands toward the posterior (Fig 1, labeled D glands) were excised for whole mounting. Mammary gland tissue was carefully trimmed away from the skin, moving as far dorsally as possible. One abdominal D mammary gland was immediately processed by formalin fixation and paraffin embedding (FFPE), while the other was whole mounted and processed as outlined below.
Whole Mount Preparation:
Immediately following tissue resection, mammary glands were stretched across an electrostatically charged slide and submerged in room temperature, 10% neutral buffered formalin overnight for fixation. Slides were removed from formalin and placed into an acetone bath at room temperature overnight to de-fat. Overnight may not be enough time to entirely remove all fat; however, overnight is sufficient and allows for sooner and more visible staining. Mammary glands will become translucent and may require up to 3 days in acetone to remove fat, depending on the thickness of the mammary gland.
Following defatting of the mammary glands, slides were submerged in aluminum carmine staining solution. Aluminum carmine solution was prepared by dissolving 1g of carmine alum and 2.5g of aluminum potassium sulfate in 500 mL of RNase-free water. Boiling 20 minutes was used to completely dissolve carmine into solution. Carmine solution was refrigerated for storage and reused after filtering to remove buildup of oil remaining in mammary glands. Staining may take 1-2 days depending on mammary gland thickness. Glands were considered fully stained when white tissue was no longer visible through the back of the slide. Whole mounted fixed tissue not used for the purposes of RNAscope analysis can be stored long-term submerged in mineral oil.
Mammary gland tissue processing, embedding, and sectioning:
To evaluate morphological structures and lesions within carmine stained mammary gland tissue whole mounts, such as hyperplasia and ductal carcinoma in situ (DCIS) (Fig 2), regions of interest were excised from whole-mounted glands using a surgical scalpel. Excised tissue was placed in histology cassettes and processed using a standard tissue processor. To properly embed tissue in paraffin blocks, tissues were oriented such that the flat side, originally against the glass microscope slide, faced the bottom of the paraffin mold. Paraffin embedded tissues were stored at 4°C until ready to section. Messenger RNA is stable in paraffin blocks for effective use in RNAscope for up to 1 year .
Sectioning of FFPE blocks was done no more than one day prior to a planned RNAscope assay, as mRNA remains more stable within paraffin blocks than tissue sections . Prior to sectioning, paraffin blocks were placed at -20°C for a minimum of 30 minutes to improve sectioning and reduce wrinkling of tissue sections. Tissues were cut into 4 mm sections and placed onto superfrost plus microscope slides. Other slide types, suitable for RNA in situ hybridization assays, may also be used. Slides were dried fully at room temperature overnight. Depending on tissue size, it was possible to fit up to three sections per slide. This served to analyze multiple genes or controls on a single slide in an RNAscope assay.
Hematoxylin and Eosin (H&E) staining of sectioned mammary glands:
An H&E stain on sectioned tissues was used to assess tissue sample quality and to confirm the lesion of interest was present in the section. Slides were deparaffinized and rehydrated using 3 separate xylene washes followed by single washes each in 100% ethanol, 90% ethanol, 80% ethanol, and DI water. Tissues were stained with hematoxylin for 3 minutes, rinsed under running DI water for 3 minutes, soaked in bluing reagent for less than a minute, and again rinsed with DI water. Slides were dipped a few times in 100% ethanol to eliminate water before staining in eosin-Y for 30-45 seconds. Tissue was then dehydrated using washes of 90% ethanol, 100% ethanol, and 3 separate xylene washes. Samples were air dried and coverslipped using permount or similar mounting media
In this report, RNAscope assays were performed on pairs of D mammary glands, one immediately processed by FFPE and the other whole mounted, defatted, carmine stained, and processed and paraffin embedded later. The RNAscope 2.5 HD Detection Reagent-RED kit (Advanced Cell Diagnostics #322360) was used according to manufacturer’s protocol except for modifications to minimize harsh pre-hybridization treatment conditions on fragile mammary tissue. Slides were deparaffinized in two xylene, followed by two 100% ethanol washes. A hydrophobic barrier was drawn around tissue sections with an ImmEdge pen. Sections were blocked with H2O2 for 10 minutes at room temperature. Slides were submerged in target retrieval solution at a temperature just below boiling for 10 minutes, rather than the manufacturer recommended boiling for 15 minutes. Protease plus solution was added to slides and incubated in HybEZ oven (ACD Bio) at 40°C for 10 minutes, rather than the manufacturer recommended 30 minutes. Target probe was added and allowed to hybridize for 2 hours at 40°C in the HybEZ oven. Serial steps to amplify signal involved the following solutions and conditions: Amp1 for 30 minutes at 40°C, Amp2 for 15 minutes at 40°C, Amp3 for 30 minutes at 40°C, Amp4 for 15 minutes at 40°C, Amp5 for 30 minutes at room temp, Amp6 for 15 minutes at room temp, and detection reagent for 10 minutes at room temperature. Slides were washed in wash buffer between each probe hybridization and amplification step. Slides were counterstained with 1:1 Gill’s hematoxylin and water, allowed to air dry, and coverslipped with mounting media. Slides were imaged on an Aperio ImageScope CS2.
Expression Quantification and Statistics:
Representative images were selected from n=6 mammary carcinoma and n=6 non-diseased mammary tissues that were either immediately FFPE processed or whole mounted prior to FFPE processing. Mean probe per cell was quantified for DapB, Mier3, and Polr2a expression using the HALO-ISH Quantification software from Indica Labs. Two independent DMBA groups were used to provide mammary gland or mammary carcinoma tissue, each tissue type was analyzed separately by Two-Way ANOVA. Independent variables to compare mammary gland and carcinoma expression levels were processing method and RNAscope probe. Probe per cell was the dependent variable. Tukey’s post hoc test was used protect for multiple comparisons. Statistical analysis was performed using GraphPad Prism version 7.00 for Windows, GraphPad Software.