[Histonet] Fixed frozen non-paraffin mouse brain

[John Kiernan]

Dear Ed,

Adequate fixation is important. Formaldehyde penetrates quickly but reacts slowly with proteins. 4% formaldehyde made by depolymerizing paraformaldehyde (an insoluble high polymer) is the same as formaldehyde made by 10X dilution of formalin (a mixture of soluble low polymers).

For cryoprotection the sucrose must thoroughly penetrate the fixed specimen, which must sink in the concentrated solution.

Your Swiss cheese artifact is due to slow freezing. In badly frozen brains the ice crystal holes are as big as large neurons and the tissue architecture is destroyed.

Unless specimens are really tiny and frozen super-fast (special methods for electron microscopy), ice crystals always form and show later as holes in the sections. You get bigger holes with bigger specimens and slower freezing.
With very fast freezing, a skilled technician can get good cryostat sections even of small unfixed specimens such as muscle biopsies. These are needed for enzyme activity histochemistry methods used in diagnostic pathology and in research.

Cryoprotection of fixed specimens slows the growth of ice crystals. With luck, the holes are too small to interfere with light microscope studies of sections. In neuroscience research, quite thick frozen sections of samll animals' brains have been the norm for more than 50 years.

About 20 years ago I wrote a chapter that gave some quite detailed instructions and explanations, with references. (Don't do anything important just because a chapter or a review says so; check at least some of the refs!)   Here is the reference.

Kiernan, J. A. 2002. Freezing and fixation. Chapter 8 in Microscopy and Histology for Molecular Biologists. A User's Guide, ed. Kiernan, J. A. & Mason, I. G. pp. 103-143.  London: Portland Press.  ISBN 1855781417.

Your university's library in Urbana might have the book. It's out-of-print with its publisher. There are used copies on the web for much less than the original price.

John Kiernan
Emeritus neuroanatomist and histochemist
London, Canada
https://www.schulich.uwo.ca/anatomy/people/bios/emeriti/kiernan_john.html
= = =
________________________________
From: Roy, Edward J via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 04 July 2020 20:08
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Fixed frozen non-paraffin mouse brain

As a research lab, we sometimes would like to use paraformaldehyde-fixed but non-paraffin embedded tissues; paraffin embedding alters antigens and necessitates antigen retrieval, but simple fixation does not. We have done the traditional 30% sucrose before OCT and freezing, with cryostat sectioning, but results are inconsistent, sometimes producing Swiss-cheese brains. Does anybody have an alternative to 30% sucrose that is more reliable?  I didn’t see anything in the Archives after a search for “30% sucrose”.
Thanks very much,
Ed Roy

Edward J. Roy, PhD
Professor Emeritus
Department of Molecular and Integrative Physiology
University of Illinois at Urbana-Champaign
Urbana, IL 61801
217 333-3375


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