Frozen Section Technique in the Animal Research Setting
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.
KeywordsPerfusion 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
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