RE: [Histonet] formalin fixing
|From:||"Charles W. Scouten, Ph.D." |
You should check the archives for a recent discussion on fixation.
Formalin works by crosslinking proteins, wherever it finds them. The result is not subject to normal protein breakdown and decay processes. Formalin penetrates to everywhere in the tissue, albeit slowly. Anything that can be done to speed up its penetration should (ie. perfusion). Heat will not speed up penetration, but may speed up decay.
Shrinkage due to formalin can be prevented. When formalin arrives at the cell wall, the first thing fixed is the ubiquitous sodium pump proteins on the outer wall. As a consequence, sodium rushes in down the gradient from the extracellular fluid, and carries in water to maintain tonicity. Cells first swell and fill the extracelllar space, to abut neighboring cells, then proteins in the membranes are crosslinked between cells. When equilibrium is reestablished, the cells shrink and pull their neighbors in with them. The shrinkage due to formalin is thus loss of extracellular space that used to be there.
Replacing sodium in the extracellular fluid, while maintaining tonicity, will prevent shrinkage, and give unshrunk formalin fixed soft tissue. Perfusion with ordinary sucrose at 5%, as the prewash, pressurized if brain is to preserved, will fulfill this requirement. For an instrument to assist in doing this, see the following link:
Charles W. Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300
FAX 314 522 0377
From: Shibaji Shome [mailto:firstname.lastname@example.org]
Sent: Tuesday, November 04, 2003 1:37 PM
Subject: [Histonet] formalin fixing
Stupid question of the day:
I would like to know how formalin fixation works. Does formalin enter
the extracellular space and replace water? does it enter both extra and
intracellular space? The shrinkage of tissue on formalin fixing is
indicative of water loss and/or collapse of intracellular/extracellular
space? Any information, pointers to references will be greatly
appreciated. Details on working principles of formalin fixation will
greatly, vastly appreciated.
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