Re: Mitochondria

From:"J. A. Kiernan"

jim wrote:
> I've read about the Acid-haematin technique being one of the best techniques
> for staining mitochondria. The procedure asks for frozen sections of
> formal-calcium fixed material.
>   ...   I'd like to know, is the formal-calcium
> fixation and frozen sections only required if you are using the procedure to
> demonstrate phosphatidyl cholines. But for the demonstration of mitochondria
> these two requirements are not important.
The calcium salt is added to the formaldehyde fixative
to improve preservation (immobilization) of phospholipids.
The lipids get permanently immobilized and complexed
with chromium in the first step of the staining method.
The mitochondria are made largely of membrane, and are
therefore rich in phospholipids. If you fix in, let us
say, a neutral buffered formaldehyde solution, the
mitochondria will still be well preserved but their staining
by the dichromate-acid haematein method may be a little
less intense than after a formal-calcium type of fixation.
Dehydration without the dichromate treatment will remove
all the phospholipids except those that are strongly
bound to proteins - as in myelin and to a less extent
in mitochondria.

>                            ...   if not what
> other histochemical techniques demonstrate mitochindria in
> formalin fixed paraffin embedded material.

If the specimens are already embedded in paraffin,
and were not first treated with potassium dichromate,
I don't think there's a histochemical method that you
can use to stain mitochondria. There are classical
staining methods, however, such as Altmann's
picro-fuchsine, iron-haematoxylin and luxol fast
blue. You won't get the best out of these, however,
if the fixative did not contain either osmium
tetroxide or pot. dichromate. The subject was briefly
but well reviewed by HC Cook 1974: Manual of
Histological Demonstration Techniques. London:
Butterworths, p. 35-37.

A more modern approach might be to stain
immunohistochemically for a protein that occurs
only in mitochondria - cytochrome oxidase
perhaps. I don't know what's commercially available
in the way of antibodies to such enzymes.

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

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