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Perfusion Two™ Automated Pressure Perfusion
Product: #39471005
Product Overview:
Automating the perfusion system gives much greater consistency and more reproducable perfusions, as well as greater convenience and ease of operation. The Perfusion Two complete system comes with an automated air compressor that quietly pumps 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.
This instrument enables an animal sacrifice perfusion procedure that allows:
• Fixation without shrinkage
• Preservation of the extracellular space
• Thorough wash out of red blood cells
• Stronger labeling of cell contents with less background.
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.
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 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 backets for wall mounting.
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.
Journal Citations
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Preservation of the Extracellular Space During Fixation of the Brain For Electron Microscopy. Cragg, Brian. Tissue and Cell 12: 63-72, 1980
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Artifacts in routinely immersion fixed nervous tissue. Garman, RH. Toxicol Pathol 1990;18(1 Pt 2):149-53
In order to demonstrate the types of artifacts commonly seen in inadequately fixed central nervous system (CNS) tissues, rat brains were fixed by either perfusion or immersion in 10% neutral buffered formalin. Perfused brains were either removed immediately or left in situ for 1 hr prior to removal. Some of the brains destined for immersion fixation were first allowed to sit for either one-half or 1 hr in either room air or in an isotonic saline solution. Other brains were subjected to various handling procedures immediately after removal from the cranial vault in order to simulate the types of trauma which commonly occur during routine removal/dissection. Commonly observed artifacts included the presence of basophilic (dark) neurons, retraction spaces around neurons, vessels, and glial cells, displacement of the neuropil, and neuropil vacuolar change. The results of the fixation and handling procedures utilized in the collection of these brains substantiate the well documented fact that the least degree of artifact will be seen in brains fixed by perfusion and left in situ for a reasonable period of time (several hours) prior to removal.
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Post mortem origin and mechanism of neuronal hyperchromatosis and nuclear pyknosis. Jan Cammermeyer. Experimental Neurology Volume 2, Issue 4, August 1960, Pages 379-405.
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Perfusion Fixation of Research Animals. Scouten, CW, R. O'Connor and M. Cunningham. Microscopy Today 14: 26-33, 2006
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A Practical Guide to Frozen Section Technique Peters, (ED), Chapter 9, Scouten, Charles " Frozen Section Technique in the Animal Research Setting" Springer, 2009
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Extracellular Space Volume Measured by Two-Color Pulsed Dye Infusion with Microfiberoptic Fluorescence Photodetection. M. Magzoub, H. Zhang, J. Dix, A. Verkman. Biophysical Journal, Volume 96, Issue 6, Pages 2382-2390
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Effects of excitotoxic thalamic intralaminar nuclei lesions on attention and working memory. Lori A. Newman and Joshua A. Burk. Behavioural Brain Research
Volume 162, Issue 2, 30 July 2005, Pages 264-271
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An in vivo microdialysis assessment of concurrent MDMA and cocaine administration in Sprague–Dawley rats. John J. Panos and Lisa E. Baker. Psychopharmacology, Volume 209, 2010
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An assessment of methods for aligning two-dimensional microscope sections to create image volumes. R. Beare, K. Richards, S. Murphy, S. Petrou and D. Reutens. Journal of Neuroscience Methods Volume 170, Issue 2, 30 May 2008, Pages 332-344
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Attentional demands for demonstrating deficits following intrabasalis infusions of 192 IgG-saporin. Joshua A. Burk, Matthew W. Lowder and Kathleen E. Altemose. Behavioural Brain Research Volume 195, 2008, Pages 231-238.
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Systemic and intrabasalis administration of the orexin-1 receptor antagonist, SB-334867, disrupts attentional performance in rats. Karen E. Boschen, Jim R. Fadel and Joshua A. Burk. Psychopharmacology, Volume 206, Number 2 / October, 2009