Alessandra,

As usual, John is right on the money!

Appropriate, complete fixation is crucial for the demonstration of
mitochondria.

I would also recommend Baker's acid hematin for straight LM.
However, the Altmann - Metzner stain is absolutely brilliant on paraffin
sections. I still have the slides I stained (Gulp) 45years ago!
There is one added bonus to this technique for your purposes. The Acid
Fuchsine used in Altmann's is stunningly fluorescent by confocal microscopy.
In addition, the photobleaching is negligible.


Bryan R.Hewlett



----- Original Message -----
From: "J. A. Kiernan" 
To: "Alessandra Livraghi" 
Cc: "HistoNet Server" 
Sent: Friday, October 25, 2002 12:03 AM
Subject: Re: Mitochondrial immunostaining (long)


> Alessandra Livraghi wrote:
> > Here I am again. Some time ago I was asking for some histochemical
method to
> > label mitochondria in  human tissues...I got some good advices and the
> > staining with SDH and NBT works pretty well.
> > Now I'm back on the same issue, but I'm looking for some GOOD advices
about
> > antibodies to stain mitochondria in paraffin embedded and/or frozen
sections
>
> The very brief (4 min) formaldehyde treatment of your
> cryosections will not fix anything, and the 100% alcohol
> that follows will wreck the organelles. Mitochondria are
> fragile little things, destroyed or deformed by some fixatives
> and destroyed or deformed by water etc if you don't fix them.
>
> Dehydrogenase histochemistry on unfixed or almost unfixed
> sections certainly shows mitochondrial enzyme activity, but
> the sites of deposition of the stain do not necessarily
> correspond to the mitochondria that you'd see in an electron
> micrograph or in a cell vitally stained with janus green B or
> one of the modern vital fluorochromes, or with a classical LM
> stain for properly fixed mitochondria. These statements apply
> equally to immunostained mitochondrial antigens in inadequately
> fixed cells.
>
> The best fixatives for mitochondria in light microscopy are
> archaic mixtures containing potassium dichromate and osmium
> tetroxide applied to minute fragments of tissue, and thoroughly
> washed out before processing into paraffin. Manfred Gabe, who
> was an expert, wrote in his 1976 book that the clearing agent
> should be cedarwood oil (which is viscous and not all removed
> during infiltration with wax). I don't know what all this does
> to the antigens; probably noone has tried to find out.
>
> A neoclassical approach is to fix in neutral formaldehyde (added
> calcium ions improve the picture) or glutaraldehyde (or a mixture
> of the two), and post-chrome the specimen. After some days in an
> aqueous dichromate solution the hydrophilic phospholipids (those
> of mitochondria and myelin) are rendered insoluble in organic
> solvents and therefore stainable in paraffin sections.
>
> I have used Bensley's copper-haematoxylin and Baker's acid
> haematein for this purpose, and I recommend the latter because
> it's easy to do and is a thoroughly investigated and fairly
> well understood histochemical method.
>
> I've never tried Altmann's stain, but have been told that the
> bright red mitochondria on a pale yellow background are
> impressive. Baker's acid haematein gives blue mitochondria
> with a dirty-yellow background. The  yellowy background is
> due to haematein acting as an anionic dye in its own right,
> without any "mordant" metal ions in the staining solution. The
> blue color is that of the complex of haematein with the bound
> chromium in the mitochondrial lipids. It's much the same
> colour as the aluminium-haematein complex in cell nuclei in
> sections stained with H & E (or is it H&E as a single wordlet?).
>
> There's plenty of published work in the field of mitochondrial
> staining. If you have access to a library I'll be happy to
> provide references to books, peer-reviewed original papers and
> review articles in this field.
>
> --
> -------------------------
> John A. Kiernan
> Department of Anatomy and Cell Biology
> The University of Western Ontario
> London,   Canada   N6A 5C1
>    kiernan@uwo.ca
>    http://publish.uwo.ca/~jkiernan/
>
>