This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.
As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.
Postnatal mammary gland development and ductal morphogenesis are processes largely influenced by hormonal signaling at the onset of puberty. In mice and rats, commonly used model organisms of mammary gland biology, this process begins around 3 weeks of age, where rapid proliferation and differentiation result in the formation of the mature parenchyma. The mature mammary gland can undergo numerous rounds of expansion and involution, a property that has been under investigation since the early 20th century. Within the context of hyperproliferation and cancer development, mammary gland transplantation techniques were developed in the 1950s1, and enhanced by the quantitative methodology contributed by Gould et al. in 19772,3,4. Refinement of the transplantation technique in rodents has contributed to major advances in understanding normal mammary gland biology that are still widely used to study the effect of various treatments and genetic manipulation on normal mammary gland development and disease states.
Many hypotheses have been generated and subsequently tested using mammary gland transplantation, first described by DeOme et al. in 19591. Experiments across several decades showed the propensity of ductal tissue excised from donor mammary glands to repopulate the entire fat pad5,6,7 and indicated that a critical component of mammary gland development resides in these epithelial structures. Later studies in mice showed that a single mammary stem cell can repopulate a cleared fat pad and contributed to the discovery of a single, common progenitor of basal and luminal mammary epithelial cells8,9,10. In line with these conclusions, it has been suggested that transplantation increases the pool of cells with multilineage-repopulating potential as a result of plasticity, allowing the grafted cells to grow a functional mammary gland7,10,11,12,13. Importantly, the use of transplantation techniques in rodents overcomes the limitations of cell culture-induced abnormalities14 and often provides results in just a matter of weeks.
While the procedure was originally described in the context of preneoplastic lesions in mice, it was soon expanded to rats and used in conjunction with the carcinogen treatment to establish multiplicity as a measure of cancer susceptibility15, but the popularity of transplantation techniques has followed the development of genetic tools for each species. Although mouse studies incorporating transplantation have contributed many translational findings, the parenchyma of the rat mammary gland resembles the human more closely16,17 and offers distinct advantages for studying estrogen receptor-positive (ER+) breast cancer. Mammary tumors are inducible in both species, but they differ in terms of hormone sensitivity and gene expression profiles. A primary difference is that rat mammary tumors express and depend on the function of ovarian and pituitary hormone receptors, namely, estrogen and progesterone (PR), similar to the luminal-A subtype of human breast cancer. Indeed, mammary epithelial cell transplantation, as described in this protocol, has been used to study genetic variants involved in breast cancer and determine the cellular autonomy of effects on mammary epithelial cells18.
In addition to the tumor biology, the ductal epithelium of the normal rat mammary gland exhibits a higher level of branching and is flanked by a thicker layer of stroma than the mouse. The importance of the stroma is well-documented in mammary epithelial transplantation studies. Mammary epithelium must interact with fatty stroma, and ideally its own mesenchyme, to undergo its characteristic morphogenesis19,20. Grafting tissue into a recipient mammary gland provides an optimal environment; however, the presence of endogenous epithelium can interfere with results. Preclearing the mammary gland of endogenous epithelium is commonly performed in mouse transplantation assays and requires surgical excision of endogenous mammary tissue and/or removal of the nipple1,21,22. Although possible, preclearing the mammary epithelium in post-weanling rats is not as widely-performed, mainly due to the ineffectiveness of clearing the growing mammary tree in post-weanling rats. Since it has been shown that regions of adipose tissue elsewhere in the body could support the growth of transplanted mammary epithelium21,23,24, the process of preclearing can be easily avoided in rats by grafting tissue into the interscapular white fat pad.
The transplantation method described in this paper involves the injection of enzymatically dissociated mammary gland organoids (fragments of mammary ductal epithelium and other cells types capable of morphogenesis) or monodispersed cells into the interscapular fat pad in inbred, isogenic or congenic strains of laboratory rats2. Because the interscapular fat pad is normally devoid of mammary tissue, it provides a suitable environment for multiple transplantation sites without the need to pre-clear endogenous epithelium. As a result, the host animal's endogenous, abdominal-inguinal mammary glands are not subject to surgical manipulation, develop normally, and cannot interfere with interpretation of results. Additionally, the intact mammary glands can be used for comparison to evaluate host versus donor effects on the mammary epithelium development and tumorigenesis18,25. Although repopulation of the mammary gland from a single stem cell is available for mice, it has not yet been developed for rat, mainly due to the lack of availability of antibodies to select for rat mammary stem cells25,26,27. Despite this, transplantation of monodispersed mammary epithelial cells to quantify repopulating potential can be successfully performed, and those cells will develop normally when grafted into the appropriate framework2,3,4. While organoids are good for many purposes, monodispersed cells are required for quantitative applications, for example, to determine the number of mammary epithelial cells required for the cancer initiation following ionizing radiation treatment28 or for comparing characteristics of flow cytometrically selected mammary epithelial cell populations29.
To date, the procedure described here is the most robust method of performing mammary gland transplantation in the rat with an overall goal of studying mammary gland development and mechanisms underlying breast cancer development. Often, the donor and/or recipient animals are exposed to different variables before, during, or after the epithelial transplantation. Examples include single gene studies involving chemical carcinogenesis30, radiation28,31,32, genetic manipulation of host/donor genome18, and hormonal manipulation12. A major advantage of the enzymatic dissociation described in this protocol is the opportunity to isolate epithelial organoids or monodispersed cells for complementary experiments involving flow cytometry, 3-D culture, gene editing, and more. Future applications of this technique will include additional manipulation of donor and/or host tissue with genetic engineering. For example, donor cells can be genetically altered ex vivo at any chosen genomic locus using the CRISPR-Cas9 gene editing system. Similarly, recipient rats can also be genetically altered to study the interaction between donor and recipient engineered genetic factors.
All animals were housed and maintained in an AAALAC-approved facility, and experiments described in this protocol were approved by the MUSC Institutional Animal Care & Use Committee (IACUC). Animals for use in reciprocal transplantation should be an inbred or isogenic strain, with congenic status preferred or backcrossed for at least 6 generations.
1. Harvesting donor rat mammary gland epithelium
2. Extract brain tissue from euthanized donors
3. Digestion and processing of mammary gland extracts
4. Transplantation procedure (recipient rats 4-5 weeks of age)
5. Assessment of epithelial outgrowth
Donor and recipient mammary glands
The steps to isolate and prepare rat mammary epithelial cells for transplantation are shown in Figure 1A. At 4 weeks of age, the endogenous mammary gland of the donor rat has begun maturation and epithelium can be visualized on whole mounted slides stained with alum carmine (Figure 1B). One donor rat at this age will provide approximately 1 x 106 cells for transplantation. If the amount of donor tissue collected or subsequent mincing is insufficient, the yield of cells after collagenase digestion may be low. As such, it is important to collect as much mammary gland tissue as possible from the donors. Fully digested mammary gland tissue should have an oily appearance, with no visible pieces of tissue in the suspension (Figure 1C). Complete digestion of the mammary gland tissue from a single donor should result in the formation of a visible pellet that contains the mammary organoids, as shown in (Figure 1D). If a pellet is not visible, the assay may require optimization. In Figure 2, rat mammary gland and interscapular fat pad locations are shown. Results cannot be interpreted unless biological reference points are properly identified at the time of tissue collection (Figure 2A) and transplantation (Figure 2B). In this assay, the medial blood vessel of the interscapular white fat pad is used as a biological reference point.
Qualitative and quantitative assessment of epithelial outgrowth
The presence, absence, or abundance of epithelium can be evaluated to determine success of the experiment, as well as the autonomy of effects related to experimental variables. For the latter, certain studies may require whole mounted slides with the endogenous abdominal-inguinal mammary gland of the host for comparison. As a preliminary measure, light microscopy can be used to document the epithelial outgrowth as a binary outcome. These data can be statistically analyzed to test the hypothesis that graft rejection is dependent on donor or recipient variables. A reciprocal transplantation experiment where each of these factors has 2 levels- for example, wildtype (WT) vs knockout (KO), will create 4 transplant groups for hypothesis testing (Figure 3A). The transplant groups, expressed as donor:recipient genotype, are: WT:WT, WT:KO, KO:KO, KO:WT. When isogenic or near-congenic animals are used, graft rejection is minor and occurs equally across the transplant groups. One advantage of multiple sites for transplantation within the interscapular fat pad is a reduction of recipient animals needed, since 2 donor cell types can be evaluated in a single host. Additionally, both sites can be used to test a single donor cell type at a higher incidence rate, using the same number of rats. Using genotype as an example, this is demonstrated in Figure 3B.
The epithelial outgrowth can also be analyzed using images of the slides that were previously acquired. An example of a whole mounted interscapular fat pad containing epithelial outgrowth at both graft sites (recorded as positive outcomes) is shown for an experiment using the mammary cell transplantation protocol described here (Figure 3C). Lack of epithelium in the interscapular fat pad across many samples may indicate a technical problem with the procedure and is treated as a negative outcome.
The outcome of reciprocal transplantation experiments can be further used to distinguish effects that are autonomous or non-autonomous to mammary epithelial cells. To test the hypothesis that an effect is driven by processes in the mammary epithelial cells (cell-autonomous) or influenced by the host/microenvironment (non-autonomous), concordance of donor cell phenotype to that of the host (endogenous) phenotype is treated as a dichotomous outcome. In an example such as a carcinogenesis assay, tumor incidence can be analyzed as binary response data, and logistic regression analysis used to determine if the donor, host, or donor-host interaction contributes significantly to the tumor incidence rate at the transplant site. If the effect is driven by properties of the donor epithelium, a similar transplantation outcome can be observed across recipient groups, irrespective of the host's condition. If donor epithelium develops as if it were endogenous to the host, due to a contribution of the host's genotype or treatment, the effect may be non-autonomous. In both situations, the transplantation groups where donor epithelial cells matched the host (self:self) should be interpreted as controls, and conclusions supported by statistical analyses.
To demonstrate results of autonomous and non-autonomous effects on transplanted epithelium, an illustration has been provided (Figure 4A), along with slides from reciprocal transplantation of wild type and Cdkn1b knockout rat mammary epithelium experiments. Results of this study suggested non-mammary cell-autonomous effects18 (Figure 4B). For reciprocal transplantation outcomes classified as a binary response, the likelihood of the outcome (e.g., concordance of phenotype to host epithelium, or successful outgrowth in quantitative assays) being dependent on categorical variables (e.g., donor or recipient genotype) can be tested by building a logistic regression model for main effects and interaction terms.
Figure 1: Preparation of donor mammary glands. (A) Overview of the procedure to extract mammary gland tissue from donor animals and recover organoids for transplantation. (B) Whole-mounted endogenous abdominal-inguinal mammary glands of a 4-week old rat (typical age of transplant donors), after alum carmine staining. At 4 weeks of age, the mammary epithelium was in the process of expanding, but the ductal tree does not fully penetrate the fat pad, as evidenced by proximity to the central lymph nodes. (C) The consistency of the minced mammary gland is shown after chopping (pink) in a 60 mm dish on ice, with an appropriate amount of DMEM/F12 media to keep it moist. The adjacent images provide a comparison of the minced epithelium after transferring the slurry to Collagenase Digestion Media and when the digestion is complete, 90-120 minutes later. (D) The pelleted epithelial organoids and layer separation are visible after centrifugation. Please click here to view a larger version of this figure.
Figure 2: Identification of boundaries for tissue collection and mammary epithelial cell injection. (A) Locations of rat mammary glands for tissue collection are shown. The approximate location of endogenous abdominal-inguinal mammary glands harvested from donors and recipients is outlined. (B) After shaving and making a superficial incision, the white interscapular fat pad of a transplant recipient is exposed. Top image: the medial blood vessel (yellow arrow) is visible in the center of the incision. Bottom image: the skin on one side of the incision is lifted to show the width of the IS fat pad underneath the skin, relative to the medial blood vessel (yellow arrow). Donor epithelium is injected underneath this flap into the fat pad. Please click here to view a larger version of this figure.
Figure 3: Reciprocal transplantation schema. (A) Typical experimental design for reciprocal transplantation is shown, using genotypes as an example. Testing a single experimental variable, such as gene knockout (KO) relative to wildtype (WT) donor epithelium, creates 4 transplantation recipient groups. (B) Example design for a single recipient animal receiving 2 injections of donor material. Multiple sites for grafting are accessible when using the interscapular white fat pad because of the presence of a blood vessel along the midline. Separate preparations of wild type and knockout donor epithelium (or other test conditions) can be injected to the left and the right of the blood vessel. (C) Representative whole mounted IS fat pad tissue from a transplant recipient is displayed in the same orientation as in the example presented in (B). Mammary epithelial outgrowth is visible at two sites of transplantation on a whole-mounted slide with alum-carmine stained tissue. The interscapular fat pad was excised as a single piece 6 weeks after transplantation. Additional time for epithelial development may be needed but may also facilitate overgrowth and difficulty distinguishing individual donor grafts. Common biological artifacts may be visible: MS = muscle, TP = transplanted epithelium, BF = brown adipose fat tissue. Please click here to view a larger version of this figure.
Figure 4: Mammary cell autonomous and non-autonomous results analysis. (A) Simulation of results that may be observed when there are significant contributions of autonomous and non-autonomous effects on the phenotype of donor epithelium. In this example, the endogenous abdominal-inguinal glands from the host are used as a comparison. Concordance of the phenotype of donor epithelial outgrowth, as compared to the endogenous gland, can be used as a reference, but should not be used to exclusively determine effects. (B) Donor mammary epithelium outgrowth is shown at the site of transplantation, adjacent to images of the endogenous gland for all 4 groups in a reciprocal transplantation experiment. Non-autonomous effects on mammary epithelium observed following knockout of a single gene, Cdkn1b, suggesting the host's microenvironment affects the developing rat mammary gland18. Scale bars represent 5 mm. Please click here to view a larger version of this figure.
Step Needed | Items on ice | Thawed/Pre-warmed | Items near surgeon |
1. Preparation of mammary gland epithelium | 60 mm dish | Sterile surgical tools | |
Aliquot of DMEM/F12 | 70% Ethanol | ||
2. Donor brain extraction | Aliquot of media in 15 or 50 mL tube (approx. 1-2 mL/donor) | Balance for weighing brain, within sterile field | |
Labeled 15 mL tube for each donor, suitable for homogenization | Foil | ||
Pipette and tips (1000 µL, or electronic with 5 mL serological pipettes) | |||
Mechanical homogenizer | |||
3. Enzymatic digestion of donor glands | Sufficient DMEM/F12 for washes | Serum-free digestion media | Lab scale (g) |
DNAse I | Monodispersion Mixture | Incubator/shaker | |
Inactivation Solution | 50 mL tube (labeled) for each donor | ||
10 mL (or greater) syringe for sterile-filtering collagenase digestion media | |||
20-40 µM filters | |||
Aliquot of media to pre-wet filter(s) | |||
50 mL tube(s) for collecting filtered enzyme solution | |||
Sterile scissors for cutting disposable pipet tips | |||
Large beaker for collecting supernatant, or vacuum line for aspirating | |||
4. Transplantation | Donor epithelium + brain homogenate mixture | Hamilton syringes – 1 per donor genotype/condition | |
Aliquot of DMEM/F12 to prime syringes | Scale | ||
Sterile surgical supplies (scalpel/scissors, multiple forceps) | |||
Wound clips/sutures | |||
Gauze | |||
70% ethanol or isopropanol | |||
Beta-dine or iodine | |||
Analgesic | |||
Heat support for recipient animals | |||
Paper towels or delicate task wipes |
Table 1: Items requiring advance consideration at each step. This list is designed to be used as a reference when preparing an experiment and should not be considered exhaustive. Reagents may only be necessary for specific applications of the protocol, based on the inclusion/exclusion of optional steps. In any experiment, these items must be accessible without delay once the procedure is started.
Supplemental File 1: Serum-Free Collagenase Digestion Media Preparation. Please click here to view this file (Right click to download).
Supplemental File 2: Monodispersion Mixture Preparation. Please click here to view this file (Right click to download).
Supplemental File 3: Inactivation Solution Preparation. Please click here to view this file (Right click to download).
Supplemental File 4: Alum-Carmine Stain Preparation. Please click here to view this file (Right click to download).
This protocol describes a mammary epithelial cell transplantation technique optimized for working with rats. Isolated mammary epithelial organoids from donor rats (3-5 weeks of age) are grafted into the interscapular white fat pad of recipient rats (also 3-5 weeks of age). Results can be interpreted as little as 4-6 weeks later, using light microscopy to examine the grafted tissue; however, the optimal amount of time between transplantation and sacrifice must be determined prior to implementing a full experiment. If too little or too much time has passed, the results will neither be interpretable nor meaningful. To optimize the protocol, analyze the outgrowth in a small set of animals 6-8 weeks after transplantation. If the transplanted epithelium is present, but underdeveloped, increase the length of time. If the grafts are well-developed but overlapping features of the epithelium interfere with the analyses, consider reducing the number of weeks for epithelial outgrowth. If the amount of time cannot be shortened (e.g., in carcinogenesis experiments), it is advised to inject the same type of donor epithelium into both transplantation sites (one on each side of the medial blood vessel), as outcomes cannot be interpreted from individual sides with 100% certainty. If any type of interaction (autocrine, paracrine) is suspected, it is strongly advised to include additional control animals injected with the same type of donor epithelium into both transplantation sites. Critical steps in the procedure include proper quantification of donor cells after enzymatic digestion, and uniform mixing with brain homogenate. Extra care must be taken at these steps to ensure the number of transplanted cells is consistent across recipient animals. Also, during injection, make sure that the grafted tissue mixture is not leaking out of the interscapular fat pad. 3. Excising the interscapular fat pad at endpoint of the experiment. The entire pad can be removed as a single piece, but care must be taken if choosing to separate the 2 sides of the interscapular fat pad, cutting only after identifying the medial blood vessel. It can be difficult to determine the side from which outgrowth originated, especially when one graft has overgrown into the other, making removal as a single piece more ideal.
A common modification of the procedure is the addition of a carcinogenic treatment of the recipient rat25,29. The grafted tissue can retain the susceptibility to carcinogenesis that was possessed by the donor rat25, or, conversely, the donor tissue can adopt the susceptibility of the host30. These effects can only be determined when using the interscapular fat pad as the site of transplantation, because the endogenous mammary glands remain intact and function as a positive, internal control.
When using mammary gland organoids for transplantation, absence of epithelial outgrowth may be due to problems with the donor cell preparation or injection procedure. Graft rejection can also occur when the recipient and the donor strain are not congenic, causing an immune response in the host. In such cases, the recipient immune system recognizes the donor tissue as non-self, initiates an immune response, and the grafted tissue fails to grow. To reduce the risk of graft failure when donor and recipient are on different genetic backgrounds, a minimum of 6, and, ideally, more than 10 generations of backcrossing are recommended to prevent challenges that can affect result interpretation. At 6 backcross generations, most grafts will grow out, but a minority might still fail. When troubleshooting donor cell preparation as the cause of graft failure, consider whether the enzymatic digestion was too harsh, cells were kept at too high or too low of temperatures, sources of contamination, optimization of donor cell numbers used for the assay, or other protocol deviations affecting cell viability and outgrowth.
Single mammary stem cells have been shown in the mouse to be able to reconstitute a functional mammary gland, illustrating that the addition of hormonal support is not necessary for the primary outcome. The addition of brain homogenate significantly improves the outcome of transplantation by serving as a structural matrix for the donor cells and reducing the risk of migration transplant rejection3,34,35,36. When combined with brain homogenate, the minimum number of mammary epithelial cells required for transplantation is reduced more than 10-fold, as compared to alternatives3. Importantly, admixture of syngeneic brain homogenate has not been shown to affect the phenotype of transplanted epithelium, and has produced consistent outcomes in mammary carcinogenesis and susceptibility studies for over 40 years.
Some may argue that the interscapular fat pad is not representative of the endogenous mammary fat pad because of the anatomical distinctions: the proximity of the IS fat pad to brown adipose tissue, potential differences in blood vessel density resulting in differences in exposure to hormones or the presence of prominent lymph nodes in the inguinal-abdominal fat pad, which may expose the epithelium to different levels of cytokines. Although this has not been specifically tested in rats, both of these depots are subcutaneous and develop prior to visceral adipose37,38; in human adipose, greater molecular differences exist across adipose regions, and the heterogeneity within groups is not fully understood38,39. An additional factor to consider is that the white interscapular and mammary fat pads share Myf5+ mesenchymal precursor lineage, but differ in the number of cells derived from that population40. Notwithstanding, there is sufficient evidence to suggest the white interscapular fat pad provides a microenvironment similar to that of the lower mammary gland. Mammary epithelium recombined with its own mesenchyme develops a typical mammary pattern20, an effect that is well-document in rodent studies and supports the observations in human adipose tissue19,20,24,41,42. Above all, the primary determinants of mammary epithelial transplantation success in both rats and mice are the size and integrity of the fat pad43,44. In using this technique, many breast cancer susceptibility studies have proven that functional mammary tissue can be effectively and routinely generated when transplanted into the white interscapular fat pad18,30,45.
Because of the high compatibility, the epithelial outgrowth is amenable to mammary cell-autonomous and non-autonomous factors and will respond to hormonal manipulation of the recipient rats, for example, to promote differentiation or functional secretion of milk. Transplantation of organoids is often used to study factors that affect mammary gland development and/or carcinogenesis. Organoids can be further digested to single cell suspensions to facilitate quantitative interpretation of results. While the method described in this paper can be adapted to graft intact sections of mammary gland tissue (as is commonly performed in mice), the enzymatic dissociation steps allow more detailed conclusions to be made. Since preclearing the endogenous mammary fat pad in the rat is not feasible, this is currently the only method allowing for grafting of rat mammary epithelium.
The authors have nothing to disclose.
This work was funded by the Hollings Cancer Center's Cancer Center Support Grant P30 CA138313 pilot research funding from the National Institutes of Health (https://www.nih.gov/), and funds from the Department of Pathology & Laboratory Medicine at the Medical University of South Carolina. We would like to thank Marijne Smiths for recording the interview statements.
0.2 µM syringe filters | Fisher Scientific | 09-715G | sterile-filtering collagenase digestion media |
1.5 – 2.0 mL microcentrifuge tubes (sterile) | Fisher Scientific | 05-408-129 | containing resuspended cells and/or brain homogenate mixture |
100 µM cell strainers | Corning | 431752 | filtering brain homogenate |
100 uL gastight syringes with 25 gauge needles | Hamilton | 81001 & 90525 | For injecting graft mixture into recipient animals (1 per donor genotype/condition) |
1000 uL pipette tips + pipette | – | – | transferring cells/mixtures/tissue |
15 mL polypropylene tube | Falcon (Corning) | 352196 | brain homogenate mixture storage, or cell : homogenate mixture for transplantation |
40 µM cell strainers | Corning | 431750 | filtering organoids after washing the cell pellet |
50 mL polypropylene tubes | Fisher Scientific | 05-539-6 | for collagenase digestion of donor mammary gland tissue |
60 mm dishes | Thermo Scientific | 130181 | for mincing tissue |
Alum Potassium Sulfate | Sigma-Aldrich | 243361/237086 | staining mammary gland whole mount slides |
Anesthesia vaporizer for veterinary use | – | – | follow institutional protocol |
Beta-dine or iodine | – | – | |
Borosilicate glass culture tube for homogenization | Fisher Scientific | 14-961-26 | for homogenization of brain (use appropriate tube for homogenizer) |
Carmine | Sigma-Aldrich | C6152/1022 | staining mammary gland whole mount slides |
Cell counting apparatus | – | – | |
Clean animal cages for recovery | – | – | follow institutional protocol |
Collagenase Type 3 | Worthington Biochemical Corp. | LS004183 | enzymatic digestion of minced mammary gland tissue from donor rats |
deionized water | – | – | for chemical solutions |
DMEM/F12 | GIBCO | 11320033 | for mincing tissue, collagenase digestion media and resuspending epithelial cell mixtures |
EDTA | – | – | monodispersion mixture |
Ethanol, 200 Proof | Decon Labs | 2705/2701 | mammary whole mount slide fixative, mammary whole mount slide washes, cleaning surgical incision sites (diluted) |
Fetal Bovine Serum (FBS) | Hyclone | – | inactivation solution |
Gauze | – | – | |
Glacial acetic acid | Fisher Scientific | A38-212 | use for mammary whole mount slide fixative (1:4 glacial acetic acid in 100% ethanol) |
HBSS | GIBCO | – | monodispersion mixture |
Heating pads | – | – | follow institutional protocol |
Ice buckets (x2) | – | – | |
Incubator with orbital rotation | – | – | must be capable of maintaining 37°C, shaking at 220-225 RPM (for collagenase digestion of mammary tissue) |
Isoflurane anesthesia | – | – | follow institutional protocol |
Light microscope or digital camera | – | – | visualizing whole mounted mammary epithelium and/or acquiring images |
Mechanical homogenizer | Fisher Scientific | – | TissueMiser or alternative models |
Mineral oil, pure | Sigma-Aldrich/ ACROS Organics | 8042-47-5 | long-term storage of cleared mammary gland whole mounts |
Oxygen tanks for anesthesia vaporizer | – | – | follow institutional protocol |
Paper towels or delicate task wipes | – | – | |
Positively-charged microscope slides | Thermo Scientific | P4981-001 | mammary gland tissue whole mounts |
Postoperative analgesic | – | – | Institutional protocol |
Scale | body weight measurements of animals, proper dosing of pain medication | ||
Shaver | – | – | electric clippers, or other |
Staining jars | – | – | minimum of 1 per chemical wash, size appropriate for the number of slides, glass preferred |
Sterile field drapes | IMCO | 4410-IMC | used during transplantation |
Sterile scissors and forceps x3 (autoclaved) | – | – | autoclave surgical tools used for donors and recipients |
Syringes: 5 mL (or greater) | – | – | for sterile filtration of collagenase digestion media |
Trypsin | Worthington | monodispersion mixture | |
Waste collection receptacle for liquids (poured or aspirated) | – | – | |
Wound clip applier, clips, and removal tool | Fine Science Tools | 12020-00 | Closing the skin incision over the interscapular white pad pad |
Xylenes | Fisher Scientific | X3S-4 | clearing mammary gland whole mount slides after staining |