Standardized Post‐Mortem Examination and Fixation Procedures for Mutant and Treated Mice
A procedure for post‐mortem examination (or necropsy) of mice is provided. The aim is to obtain a “holistic” picture of organs and systems at the anatomical and histological levels. The major issue is tissue preservation, which is achieved by rapid transfer into a fixative solution, usually neutral buffered formalin. Fixation is the first of the four basic steps in histopathological analyses of tissues, which also include embedding, sectioning, and staining. The protocols provided here describe routine methods for tissue fixation, as these methods are integral parts of any necropsy procedure. There is also a Strategic Planning section that addresses the overall approach to histopathological evaluation, as well as specifics such as age and gender of the mice, cohort size, and controls. Curr. Protoc. Mouse Biol. 1:17‐53. © 2011 by John Wiley & Sons, Inc.
Keywords: phenotyping; tissue collection; histology; necropsy; pathology
Table of Contents
- Strategic Planning
- Basic Protocol 1: Necropsy for Histopathological Analysis
- Basic Protocol 2: Fixation for Routine Histopathological Analyses with Formalin
- Alternate Protocol 1: Fixation for Routine Histopathological Analyses with Bouin's Solution
- Support Protocol 1: Trimming Organs and Defining Planes of Section
- Reagents and Solutions
- Literature Cited
Basic Protocol 1: Necropsy for Histopathological Analysis
Basic Protocol 2: Fixation for Routine Histopathological Analyses with Formalin
Figure 2. Steps following necropsy and fixation in histopathological analyses of mouse tissues.
Figure 4. Template for necropsy report.
Figure 5. (A) Mouse with shaved back. (B) Mouse pinned ventral‐side‐up to cork board.
Figure 6. (A) Midline incision with skin reflected. Dissection of (B) salivary glands and (C) male preputial gland.
Figure 7. Dissection of mammary gland. (A) Insertion of scissors between the skin and the mammary gland, (B) spreading scissors to dissect the mammary gland from the skin, and (C) sectioning remaining adherences.
Figure 8. Dissection of pancreas.
Figure 9. Fixation of intestine.
Figure 10. Dissection of liver.
Figure 11. Dissection of kidneys.
Figure 12. (A) Dissection of testis, (B) urinary bladder, (C) seminal vesicle, and (D) prostate.
Figure 13. Dissection of (A) paragenital fat in the female, (B) ovaries and oviduct, and (C,D) bladder, vagina, and uterus (C,D)
Figure 14. Dissected ovaries, oviduct, bladder, vagina, and uterus.
Figure 15. Dissection of (A) adrenal glands and (B) mesenteric lymph nodes.
Figure 16. Dissection of aorta.
Figure 17. Removal of neck muscles (A,B) and view of exposed thyroid and trachea (C).
Figure 18. (A,B) Injection of fixative through trachea for fixation of lungs. (C) Lateral view of trachea with esophagus and thyroid.
Figure 19. Dissection of gastrocnemius and soleus muscles.
Figure 20. Dissection of brown adipose tissue (BAT).
Figure 21. Dissection of knee joint.
Figure 22. Incision through occipital bone for dissection of brain.
Figure 23. Removal of brain. (A) Placement for insertion of scissors tips, (B,C) splitting of the skull along medial suture, and (D) removal of skull halves.
Figure 24. Brain reflected towards anterior, showing optic chiasma and hypophysis underneath.
Figure 25. (A) Placement of brain in brain matrix and (B) position of razor blades for cutting slices.
Figure 26. Dissection of tail skin. (A) Longitudinal cut and (B) removal of spine.
|Bolon, B., Brayton, C., Cantor, G.H., Kusewitt, D.F., Loy, J.K., Sartin, E.A., Schoeb, T.R., Sellers, R.S., Schuh, J.C.L., and Ward, J.M. 2008. Editorial: Best pathology practices in research using genetically engineered mice. Vet. Pathol. 45:939‐940.|
|Brayton, C., Justice, M., and Montgomery, C.A. 2001. Evaluating mutant mice: Anatomic pathology. Vet. Pathol. 38:1‐19.|
|Carson, F.L. 1997. Histotechnology: A Self Instructional Text, 2nd ed. ASCP Press, Chicago, Illinois.|
|Costet, P., Legendre, C., More, J., Edgar, A., Galtier, P., and Pineau, T. 1998. Peroxisome proliferator activated receptor alpha‐isoform deficiency leads to progressive dyslipidemia with sexually dimorphic obesity and steatosis. J. Biol. Chem. 273:29577‐29585.|
|Cross S.S., Start R.D., and Smith J.H. 1990. Does delay in fixation affect the number of mitotic figures in processed tissue? J. Clin. Pathol. 43:597‐599.|
|Fan, C.Y., Pan, J., Usuda, N., Yeldandi, A.V., Rao, M.S., and Reddy, J.K. 1998. Steatohepatitis, spontaneous peroxisome proliferation and liver tumors in mice lacking peroxisomal fatty acyl‐CoA oxidase: Implications for peroxisome proliferator‐activated receptor alpha natural ligand metabolism. J. Biol. Chem. 273:15639‐15645.|
|Fawcett, D.W. 1994. Bloom and Fawcett: A Textbook of Histology, 12th ed. Saunders Co., Philadelphia, Pennsylvania.|
|Fox C.H., Johnson F.B., Whiting J., and Roller P.P. 1985. Formaldehyde fixation. J. Histochem. Cytochem. 33:845‐853.|
|Grizzle, W.E. 2009. Special symposium: Fixation and tissue processing models. Biotech. Histochem. 84:185‐193.|
|Harvey, M., McArthur, M.J., Montgomery, C.A. Jr., Butel, J.S., Bradley, A., and Donehower, L.A. 1993. Spontaneous and carcinogen‐induced tumorigenesis in p53‐deficient mice. Nat. Genet. 5:225‐229.|
|Huang, J., Powell, W.C., Khodavirdi, A.C., Wu, J., Makita, T., Cardiff, R.D., Cohen, M.B., Sucov, H.M., and Roy‐Burman, P. 2002. Prostatic intraepithelial neoplasia in mice with conditional disruption of the retinoid X receptor alpha allele in the prostate epithelium. Cancer Res. 62:4812‐4819.|
|Khetchoumian, K., Teletin, M., Tisserand, J., Mark, M., Herquel, B., Ignat, M., Zucman‐Rossi, J., Cammas, F., Lerouge, T., Thibault, C., Metzger, D., Chambon, P., and Losson, R. 2007. Loss of Trim24 (Tif1alpha) gene function confers oncogenic activity to retinoic acid receptor alpha. Nat. Genet. 39:1500‐1506.|
|Kim, M.J., Cardiff, R.D., Desai, N., Banach‐Petrosky, W.A., Parsons, R., Shen, M.M., and Abate‐Shen, C. 2002. Cooperatively of Nkx3.1 and Pten loss of function in a mouse model of prostate carcinogenesis. Proc. Natl. Acad. Sci. U.S.A. 99:2884‐2889.|
|Krstic, R.V. 1984. Illustrated Encyclopedia of Human Histology. Springer, Berlin, Germany.|
|Leong, T.Y. and Leong, A.S. 2007. How does antigen retrieval work? Adv. Anat. Pathol. 14:129‐131.|
|Li, M., Messaddeq, N., Teletin, M., Pasquali, J.L., Metzger, D., and Chambon, P. 2005. Retinoid X receptor ablation in adult mouse keratinocytes generates an atopic dermatitis triggered by thymic stromal lymphopoietin. Proc. Natl. Acad. Sci. U.S.A. 102:14795‐14800.|
|Lillie, R.D. 1965. Histopathologic Technique and Practical Histochemistry, 3rd ed. McGraw‐Hill, New York.|
|Liu, J.L., Yakar, S., and LeRoith, D. 2000. Mice deficient in liver production of insulin‐like growth factor I display sexual dimorphism in growth hormone‐stimulated postnatal growth. Endocrinology 141:4436‐4441.|
|Luna, L.G. 1992. Histopathological Methods and Color Atlas of Special Stains and Tissue Artifacts. Johnson Printers, Downers Grove, Illinois.|
|Maronpot, R.R., Boorman, G.A., and Gaul, B.W. 1999. Pathology of the Mouse: Reference and Atlas. Cache River Press, Vienna, Illinois.|
|McManus, J.F.A. and Mowry, R.W. 1960. Staining Methods. Histologic and Histochemical. Harper and Row, New York.|
|Mohr, U., Dungworth, D.L., Capen, C.C., Carlton, W.W., Sundberg, J.P., and Ward, J.M. 1996. Pathobiology of the Aging Mouse, Volumes 1 and 2. ILSI Press, Washington, D.C.|
|Moolenbeek, C. and Ruitenberg, E.J. 1981. The “Swiss roll”: A simple technique for histological studies of the rodent intestine. Lab Anim. 15:57‐59.|
|Morel, G. and Cavalier, A. 2000. In Situ Hybridization in Light Microscopy (Methods in Visualization). CRC Press, Boca Raton, Florida.|
|Naf, D., Krupke, D.M., Sundberg, J.P., Eppig, J.T., and Bult, C.J. 2002. The Mouse Tumor Biology Database: A public resource for cancer genetics and pathology of the mouse. Cancer Res. 62:1235‐1240.|
|Polak, J.M. and Van Noorden, S. 2003. Introduction to immunohistochemistry. Bios Scientific Publishers, Oxford, United Kingdom.|
|Popesko, P., Rajtova, V., and Horak, J. 1992. Colour Atlas of Anatomy of Small Laboratory Animals, Volume 2. Wolfe Publishing, London.|
|Prento, P. and Lyon, H. 1997. Commercial formalin substitutes for histopathology. Biotech. Histochem. 72:273‐282.|
|Renshaw, S. 2007. Immunohistochemistry. Scion Publishing, Bloxham Mill, U.K.|
|Srinivasan, M., Sedmak, D., and Jewell S. 2002. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am. J. Pathol. 161:1961‐1971.|
|Start, R.D., Layton, C.M., Cross S.S., and Smith, J.H. 1992. Reassessment of the rate of fixative diffusion. J. Clin. Pathol. 45:1120‐1121.|
|Sternberg, S.S. 1997. Histology for Pathologists, 2nd ed. Lippincott‐Raven, Philadelphia, Pennsylvania.|
|Titford, M.E. and Horenstein, M.G. 2005. Histomorphologic assessment of formalin substitute fixatives for diagnostic surgical pathology. Arch. Pathol. Lab. Med. 129:502‐506.|
|Turgeon, B. and Meloche, S. 2009. Interpreting neonatal lethal phenotypes in mouse mutants: Insights into gene function and human diseases. Physiol. Rev. 89:1‐26.|
|Werner, M., Chott, A., Fabiano, A., and Battifora, H. 2000. Effect of formalin tissue fixation and processing on immunohistochemistry. Am. J. Surg. Pathol. 24:1016‐1019.|
|Wilkinson, D.G. 1999. In situ hybridization. A practical approach. Oxford University Press, Oxford.|
|Williams, P.L., Warwick, R., Dyson, M., and Bannister, L.H. 1989. Gray's Anatomy, 37th ed. Churchill Livingstone, London.|
|The EUMORPHIA Web site, provides information about understanding human disease through mouse genetics.|
|Pathbase, a database of histopathology photomicrographs and macroscopic images derived from mutant or genetically manipulated mice.|
|Provides an electronic atlas for the anatomy and histology of the mouse.|
|Provides an electronic atlas for the histology of the mouse.|
|A workbook with a set of microscope slides that illustrate the basic tissues and organs of the body.|
|Histosearch is a search engine that searches over 20,000 Web pages from histology‐related sites.|
|StainsFile is a useful site with general information and resources for histology.|
|Information on formaldehyde fixatives at the Southwest Environmental Health Sciences Center (SWEHSC), University of Arizona College of Pharmacy.|
|The Canadian Council on Animal Care (CCAC) is the national organization responsible for setting and maintaining standards for the care and use of animals in science in Canada.|