Re: [Histonet] Will brain tissue shrink even more if stored in 70% alcoholprior to processing?

From:John Kiernan

All tissues, including brain, shrink when their water
is replaced by other liquids and then by wax. Graded
alcohols will slow down the shrinkage but will not
prevent it. The lower alcohols (weaker than 70%) also
usefully remove alcohol-insoluble inorganic solutes, 
such as sodium phosphate derived from the fixative.

In the case of liver, formaldehyde fixation reduces the 
volume to 99% of the original. The volume is 80% after 
dehydration to 100% ethanol, and 70% after clearing and 
infiltration with paraffin. [Data from JR Baker's 
"Principles of Biological Microtechnique" (1958) p.76, 
citing work by W.Berg (1908) that was quoted in 
K.Tellyesniczky's  article in R. Krause's "Enzyklopaedie 
der mikroskopischen Technik", Vol 2 (1926). Krause and
Baker are great classics in this field.] 

The reason for storage in 70% alcohol is that this
liquid is generally considered to protect specimens
from autolysis and attack by microorganisms without
making the tissue unduly hard.

If the original dimensions of your CNS specimens are
important data for your investigation, you should make
linear measurements before starting to dehydrate and
after the sections are mounted and stained, to obtain
a fudge factor for converting measurements to those
of the wet specimen. This factor will not take into
account differential shrinkage of cells, which is a
prominent artifact in paraffin sections of 
formaldehyde-fixed CNS. The empty spaces seen around 
large neurons (and often also around capillaries) form 
during dehydration, clearing and infiltration - probably
mostly in the last step, because they are not usually
seen in sections of plastic-embedded brain. 
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
CM Bush wrote:
> Dear Histonet,
> Hello, here is my first post to the list, thank you in advance for your help.
> Summary:
> Having problems with mouse and human brain tissue shrinkage durning processing, but would like to better preserve antigens, concerned storage of tissue 70% ethanol will shrink tissue even further:
> We have lots of brain specimens, stored in formalin for a long time (months up to 10 years), both human and mouse.  We perform immunohistochemistry on paraffin embedded brain.  Of course, there is the massive over fixation. Also the tissue shrinks a lot during processing.
> In my last lab, after brain tissue had been fixed in formalin for 7 days we would store the tissue in 70% ethanol.  I'd like to start storing the brain tissue I work with now in 70%, but I'm worried about the tissue possibly shinking too much, as the tissue already seems to shrink by greater than 60% after processing.
> (Previously, we would gradually increase the alcohols, 30% for 1 hour, then 50% for an hour in a bucket, room temperature on a rocker table, then put the cassettes into a bucket of 70% and store at room temp or 4C- does this process help any with controling the shrinkage factor?)
> Maybe this is a little bit long...thank you very much for your time.
> CM Bush

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