X-Message-Number: 23595 From: "Basie" <> Subject: The atmospheric oxygen is known to cause Date: Thu, 11 Mar 2004 01:40:54 -0500 "Comments Microdissecting frozen sections of mouse tissues was more effective immediately after staining than it was a few days later from the same slides. Different kinds of glass slides were tested by microdissecting mouse brain tissue. Initially, all morphologically distinct sub-populations of brain cells were lifted off, even from positively charged slides. However, when a block of frozen brain tissue was re-used (after being kept at - 70 oC between the cutting sessions), certain sub-populations were no longer picked up, regardless of the glass type. This effect was not seen when multiple thick sections of a re-used block were trimmed off, before thin sections were picked up on a slide and processed. On the other hand, smooth muscle of mouse gut was first lifted off effectively next day after staining. In case of fibrous tissues, for example heart muscle or kidney tubules, fragments of tissue torn out by the adhering plastic were irregularly shaped and larger than a field targeted with the beam. Discussion LCM appears to be a useful approach to analyze nucleic acids extracted from morphologically homogeneous clusters of cells, although fine adjustments of the available protocols may be necessary for particular types of tissue, or even particular cell sub-populations within a single organ, in case of frozen sections (brain, gut). Duration of exposure to atmospheric oxygen, before fixation as well as after staining, came into view as critical. The atmospheric oxygen is known to cause changes of physical properties of all materials over period of time; the process has been well documented for material as strong and durable as HDPE (high density polyethylene) used to make components of orthopedic implants. The fact that all cellular components of living organisms are also known to be prone to oxidative degradation, leads to a conclusion that the same process may be responsible for loss of tissue integrity, particularly in sections as thin as a few mm that were fixed with ethanol (dehydrating and denaturing agent). This would explain the observed difficulty in microdissecting frozen sections several days after staining. The smooth muscle's transferability only next day after staining can be explained by the same mechanism, i.e., gradual, oxidative tissue breakdown; initially, the tissue must have been too strong to allow tearing the targeted region out of its surrounding. In fibrous tissues, with abundant extracellular matrix, the problem with breaking at the circumference of laser beam, is manifested by tearing out fragments of the tissue larger than 30 mm and shaped irregularly, as dictated by the natural tissue architecture. Thus, successful tissue lifting off appeared to depend on tissue integrity, and various cell sub-populations seemed to deteriorate with variable rate. " Do CI and Alcor remove the air from the cryo protectant before it is infused in patients? I hope so. Basie Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=23595