Re: iron and formalin (NMR etc)

From:"J. A. Kiernan" <>

On Wed, 20 Jun 2001, Chastity Diane Shaffer Whitaker wrote:

> articles I have read .. use of formalin based 
> fixation solutions may cause iron to come out of the 
> cells/organelles/etc. ... detected by Perls' Stain ...
> ... would affect my results as well.
> ... some groups use methacarn rather than formalin ...
> Are there any  suggestions, articles for references ...

Yes, lots. The most important one for any research worker
is a major textbook of histochemistry. There are only
two in English: AGE Pearse (1980-90) and RD Lillie (1976).
Both contain references to original papers; Pearse is 
bigger and a bit more recent, but Lillie often provided 
better practical advice. (Iron histochemistry hasn't 
changed very much since the 1930s, except for some
methods more sensitive than Perls's in the 1990s.)

These books will tell you that it's acidity that extracts
iron, not formaldehyde. Before about 1965 brains and other
large specimens were often fixed in simple aqueous formalin,
which acidifies on standing. Large and valuable things like
human brains are often kept for many years because useful
research can be done with quite small pieces. Since about
1970 buffered formalin (pH 7-7.6) has been universally 
preferred for all purposes, especially storage of wet 

If your brains were initially fixed in phosphate-buffered
formaldehyde and the pH of the current storage solution
is higher than about 6 the iron deposits should, in 
principle, still be in their ante-mortem locations. 

Are you really doing NMR imaging to see where the
iron is in fixed brains?  This sort of thing has been
done much less expensively and with much better
resolution for about a century, with simple
histochemical methods (such as that of Perls, which
you mention).

John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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