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

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