The following outline is provided as a guide for maximizing the information obtained from your mouse. Since considerable information can be obtained from analysis of microscopic changes in the mammary gland, it is particularly important to give the pathologist representative "uninvolved" mammary tissue in addition to gross mammary lesions. This can give insight into the preneoplastic processes occurring in your model. Summary Table.

PROCEDURE:

1. For any newly generated mouse model, the founder mice and a select number of offspring (ideally no less than 2-3) should be subjected to a complete necropsy in order to get a complete description of the phenotype. This will allow identification of any additional phenotypic changes that might impact on mammary tumorigenesis studies. A guideline for performing complete necropsies can be found at http://www.ncifcrf.gov/vetpath/necropsy.html.
2. For mice in a mammary tumorigenesis study, it is preferable to have a minimum of 5 mice/group. The use of appropriate controls is critical. There should be an equal number of age-matched, non-engineered or untreated controls, of identical genetic background and parity. Note that mammary gland morphology and the incidence of spontaneous mammary tumors varies widely between different genetic backgrounds. This is a particularly important issue when working in a hybrid background. The pathologist should be supplied with the information about the mice found in the Pathology Request Form.
3. For a mammary tumorigenesis study, the mouse should be opened and examined grossly, with any lesions or gross changes noted on the necropsy sheet. Examine all mammary glands grossly and note positions and size of lesions on a diagram of the glands (See Figure 6.). Assign a unique post-mortem number to the mouse.
4. To maximize the amount of information obtainable from each mouse, the tissues indicated in Table 7 should be harvested for each mouse. Tissues of immediate interest should be processed to paraffin block as quickly as possible (within 48h for formalin) as this gives best results for immuno-histochemistry. Organs that are not immediately useful can be fixed and saved for later use (see #8 below). Correct identification of all mice is critical. It is useful to include the identifier (e.g. ears if mice are ear-notched) to allow subsequent cross-checking of mouse ID with PM number.
5. Mammary masses: For masses >0.5cm, it may be desirable to snap freeze half for molecular analysis and fix half for histology (see notes about fixatives below). For smaller masses, fix all for histology. If possible include some uninvolved gland, and harvest the contralateral gland for both histology and molecular analysis.
6. Whole mounts: To visualize the morphology of the ductal tree, it is desirable to whole mount some glands from a select subset of animals. The #4 and #9 (abdominal glands) are generally best for whole mounting because of the presence of the lymph node for orientation. A protocol for mammary whole mounts can be found at http://www-mp.ucdavis.edu/tgmice/HistoLab/Histolab.html. Lesions identified at whole mount can subsequently be sectioned for histology.
7. Trimming and sectioning mammary glands: For trimming mammary glands, remove mammary gland from skin and put on paper (rough DRY brown paper towels work well). Press gland on towel and flatten with forceps. Alternatively, the gland can be spread on a glass slide for better visualization. You can trim off some fat at this point. It is helpful to have a magnifying glass present when trimming muscle away from glands (esp. for thoracic glands). Glands are then fixed (see below).
8. Fixatives:
9. If the study will require use of specific antibodies for immunohistochemistry, it is critical to research the optimal fixative for the antibody of interest in advance. Remember that the mammary epithelium is embedded in excessive amounts of fat that either needs to be trimmed or may require special defatting procedures.
10. For many applications, 10% neutral buffered formalin can be used. Agitate tissues in a 10x-fold excess volume of fixative overnight at room temperature and then change to fresh neutral buffered formalin for storage. For best results with immunohistochemistry, PROCESS TO BLOCK WITHIN 48h. When saving tissues in fixative, double bag all untrimmed tissue with formalin. Recheck bags after 6 mo. and top up with formalin if necessary.
11. For in situ hybridization, best results are usually obtained with 4% paraformaldehyde as fixative. Fix for 3-5d at 4^oC. Paraformaldehyde needs to be fresh (store at 4^oC for a max of 4 days). Store fixed tissues, blocks and tissue sections at 4^oC.
12. The study and grading of nuclear atypia is better with some acidic or heavy metal fixatives (Bouin’s, Zenker’s, B-5 ect). Tissue stored in formalin can be post-fixed with these fixatives to improve nuclear detail.
13. Sectioning and staining. When cutting sections from mammary gland blocks, cut longitudinally ("fried egg morphology"). 5u sections are optimal. Hematoxylin/eosin staining is usually optimal for histology. A very light hematoxylin counterstain is generally used alone for immunohistochemistry. However, other nuclear counterstains may be better for specific purposes. Sometimes diagnosis will be aided by additional special stains (e.g. immunohistochemical staining for smooth muscle actin?).