Product Information for...
Perfusion Two™ Automated Pressure Perfusion
Product: #39471005
Product Overview:
The Perfusion Two system includes an automated air compressor that quietly pumps the air tank of the Perfusion One up to 300 mm Hg and stops. As each animal is being prepared, the pressure is set up to begin at the turn of a switch. An aperture between the air tank and the fluids provides the correct rate of pressure rise after the perfusion is started. No need to pump up the pressure, and less need for hands involved.
Automating the perfusion system gives much greater consistency and more reproducable perfusions, as well as greater convenience and ease of operation.
- Perfusion Two enables an animal sacrifice perfusion procedure that allows:
- More consistent, reproducible fixations
- Fixation without shrinkage
- Preservation of the extracellular space
- Thorough wash out of red blood cells
Depending on perfusion parameters widely varying between labs (height of gravity flow bottles, duration of flow, volume and pressure of flow) varying amounts of red blood cells remain in brain. Red blood cells autofluoresce, and catalyze HRP reactions readily, and thus contribute to background staining in many common procedures.
The classic animal transcardiac perfusion procedure with saline prewash followed by formaldehyde fixative results in an about 20% shrinkage of brain volume, and gross anatomical distortion. The extracellular space which is about 20% of brain volume in living animals is absent; under electron microscopy, cells show up butted against each other.
The shrinkage occurs when a prewash with physiological saline is followed by fixative. The first action of fixative on the cell membrane is to shut off the sodium pump proteins. Sodium rushes into the cell, followed by water to maintain tonicity, and cells swell and expand. Membrane proteins are fixed and crosslinked to neighboring cells in the swollen position. Later, the cells stabilize and contract, and pull other cells with them. The result is gross shrinkage, local distortion and torn membranes with lost cellular contents from some cells.
Tissue Shrinkage can be completely prevented if the extracellular fluid with ions is replaced by a nonionic isotonic solution that cannot enter the cells. This can be accomplished by prewash with 9.25% (isotonic) sucrose in distilled water. Start the prewash at low pressure, and pump up to 300 mmHg to break the blood brain barrier and wash the extracellular fluid (and red blood cells). Switch shortly thereafter to fixative.
The perfusion takes the same time as the traditional procedure, and accomplishes the favorable results bulleted above. An extra bottle is supplied for any post wash or cryoprotectant desired.
The Perfusion One rack is 26"W x 6" D x 15" H. and is equipped with brackets for wall mounting. Perfusion Two pump can sit on top. See the application notes for a detailed protocol, and a sample methods paragraph that may be cut and pasted (and edited) into your manuscript for publication.
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