Advertisement

Frozen Section Technique in the Animal Research Setting

Chapter

Abstract

There are significant differences between the skills required for research animal and for clinical histology. Researchers normally use sacrifice perfusion to harvest animal tissue, and need to freeze and section whole organs, sometimes in a specific orientation. Clinical histologists normally work with small samples or fragments, immersion fix, and paraffin embed.

Perfusion clears the tissue of red blood cells, which interfere with many common cell labeling methods and block capillaries to the entrance of fixative. High perfusion pressure clears red blood cells faster and more thoroughly, and achieves faster fixation. Pre-wash must be isotonic, preferably sucrose, while the fixative solution should be hypotonic, because the cytosol becomes hypotonic during pre-wash, to avoid shrinkage of soft tissue.

Tissue that is to be frozen in order to harden for sectioning must be snap frozen throughout, a greater challenge when large blocks of tissue such as whole organs are used. Liquid nitrogen will freeze faster and create a shell around the exterior of the tissue, and then the organ will crack when the interior expands due to slower freezing. Freezing tissue in a slurry of dry ice and isopentane works better for rodent brains or similar size blocks of tissue. In setting up the microtome, blade angle should be adjusted to equal the bevel on the lower edge of the knife, regardless of the method of hardening or sectioning, and regardless of the tissue being sectioned. Commercial gelatin encasement (Brain Blockers™) can provide accurate, reproducible orientation for rodent brains. Tape transfer methods provide accurate transfer from the frozen block to the slide, with all fragments in original orientation and relative position.

Keywords

Perfusion Freezing artifact Snap freezing Organ orientation Fixation Organ shrinkage Sectioning Brain blockers Peltier freezing stage Tape transfer system Cryostat Sliding microtome Isopentane Liquid nitrogen Gelatin encasements Blade angle • Swiss Cheese Artifact 

References

  1. Baker JR (1958) Principles of biological microtechnique, Methuen & Co Ltd, London, pp 37–40Google Scholar
  2. Brodbelt A, Stoodley M (2007) CSF pathways. A review. Br J Neurosurg 21:510–520CrossRefPubMedGoogle Scholar
  3. Cammermeyer J (1960) Post mortem origin and mechanism of neuronal hyperchromatosis and nuclear pyknosis. Exp Neurol 2:379–405CrossRefPubMedGoogle Scholar
  4. Cragg B (1980) Preservation of extracellular space during fixation of the brain for electronmicroscopy. Tissue Cell 12(1):63–72CrossRefPubMedGoogle Scholar
  5. Garman RH (1990) Artifacts in routinely immersion fixed nervous tissue. Toxicol Pathol 18:149–153PubMedGoogle Scholar
  6. Geiger JRP, Bischofberger J, Vida I, Fröbe U, Pfitzinger S, Weber HH, Haverkampf K, Jonas P (2002) Patch clamp recording in brain slices with improved slicer technology. Plugers Arch – Eur J Physiol 443:491–501CrossRefGoogle Scholar
  7. Green CJ (1979) Animal anaesthesia. Laboratory animal handbooks, vol 8. Laboratory Animals Ltd., London, UK, pp 123–124Google Scholar
  8. Hong WD (2008) The Allen Reference Atlas: A digital color brain Atlas of the C57BL/6J Male Mouse, Wiley, New YorkGoogle Scholar
  9. Jongebloed WL, Stokroos D, Kalicharan D, Van der Want JJL (1999) Is cryopreservation superior over tannic acid/arginine/osmium tetroxide non-coating preparation in field emission scanning electron microscopy. Scanning Microsc 13:93–109Google Scholar
  10. König JFR, Klippel RA (1967) The rat brain. A stereotaxic Atlas of the forebrain and lower parts of the brain stem. Robert E. Kreiger Publishing Co Inc, Huntington, New YorkGoogle Scholar
  11. Maser MD, Powell TE, Philpott CW (1967) Relationships among pH, osmolality and concentration of fixative solutions. Stain Technol 42:175–182Google Scholar
  12. Medawar PB (1941) The rate of penetration of fixatives. J R Microsc Soc 61:46Google Scholar
  13. Palay SL, McGee-Russell SM, Gordon S, Grillo M (1962) Fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide. J Cell Biol 12:385–410CrossRefPubMedGoogle Scholar
  14. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates, 4th edn. Academic, New YorkGoogle Scholar
  15. Rappoport SI (1976) Blood brain barrier in physiology and medicine. Raven Press, New YorkGoogle Scholar
  16. Scouten CW, O’Connor R, Cunningham M (2006) Perfusion fixation of research animals. Micros Today 14:26–33Google Scholar
  17. Short CE (1987) Principles and practice of veterinary anesthesia. Williams & Wilkins, Baltimore, p 456Google Scholar
  18. Van Harreveld A (1972) The extracellular space in the vertebrate central nervous system. In: Bourne GH (ed) The structure and function of nervous tissue, vol 4. Academic Press, New York, pp 447–511Google Scholar
  19. Van Harreveld A, Steiner J (1970) Extracellular space in frozen and ethanol substituted central nervous tissue. Anat Rec 166:117–130CrossRefPubMedGoogle Scholar
  20. Watson C, Paxinos G, Kayalioglu G (2009) The spinal cord: A Christopher and Dana Reeve Foundation Text and Atlas. Academic, San DiegoGoogle Scholar
  21. Reid N, Beesley JE (1991) Sectioning and cryosectioning for electron microscopy. Practical methods in electron microscopy, vol 13. Elsevier, AmsterdamGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Biosystems DivisionLeica Biosystems St. Louis, LLCSt. LouisUSA

Personalised recommendations