Re: Luxol Fast Blue-Holmes stain
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | Curtis King <cking_mpi@hotmail.com> |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
On Tue, 22 Aug 2000, Curtis King wrote:
> If anyone has a procedure for a combination Luxol Fast Blue-Holmes silver
> stain could you please send it to me.
Do the complete Holmes silver, with gold toning-oxalic-acid-thiosulphate
follow-up) first. (It involves two alkaline solutions, the dilute
buffered AgNO3 and the sulphite-hydroquinone developer. These could
extract luxol fast blue if that staine were done first.)
Holmes' method is very reliable once you've got it working. You
may need to vary the pH of the borate buffer and/or halve or
double the concentration of silver nitrate. Often you can get away
with using his original (1943) technique in which the only silvering
is in the dilute buffered solution, overnight at 37C. This saves
effort, black fingers and a lot of money. Otherwise, use Holmes'
final (1947) procedure, with the pre-treatment in 20% AgNO3 and
a dash of pyridine in the overnight dilute solution. (This is the
method given in most staining manuals, but the 20% AgNO3 step is
rarely necessary. The price of silver nitrate has been ridiculously
high for many years.)
Silver methods for axons work well only if you understand the
reasons for all the steps in the method, and Holmes (1943) provided
the simplest of all these methods for paraffin sections. His paper
in Anatomical Record 86: 157-187 is one of the all-time great classics
of histotechnology. Better than Bodian (1936, 1937) and Samuel (1953),
IMHO. Peters (1955) published a series of 4 papers in the Quarterly
Journal of Microscopical Science on silver staining of axons in
paraffin sections that firmly established the validity of the chemical
principles (dating from Liesegang 1911 and the scientific study of
photographic development). Little (perhaps nothing) has been added to
knowledge of this field since 1955, but many methods, new in one way
or another, have been described. I could say much more, but it
wouldn't be answering Curtis King's question!
Technical tip. Do not rinse the slides in water after the overnight
stay in very dilute, buffered silver nitrate, before their immersion
in the sulphite-hydroquinone (Bodian's) developer. If you understand
how silver methods work, you will know the reason for this. If you
don't know why you shouldn't wash away all the AgNO3 you need to
read a textbook (or Holmes 1943, cited above) before going ahead.
Silver methods for axons have a reputation for fickleness second
only to that of silver methods for neuroglial cells. In a lab that
I visit occasionally they have a framed photo of Santiago Felipe
Ramon y Cajal on the wall, scowling down at the vessels and bottles
on the bench. Wizardry is not dead. That lab now puts out publications
with the best illustrations of immunohistochemical preps that you
could ever expect to see.
Next do the complete luxol fast blue procedure. The reason for doing
it after and not before the Holmes stain is given above.
Finally, if you want the lot, counterstain with neutral red to show
neuronal Nissl substance (cytoplasmic RNA) and nuclei. Dehydrate
quite abruptly (3 X 100% alcohol), clear in xylene (X2) and apply
the coverslips with any resinous mountant.
Ideally you'll end up with black or dark red axons, many with blue
myelin sheaths, and red neuronal cell-bodies. My experience with
combining these methods is that pleasing results are obtained only
in parts of the sections, and I don't think it's worth trying too
hard. Axon (silver) plus myelin combination stains need oil
immersion to be properly appreciated, and even then they do little
more than show myelin sheaths around the larger axons.
Conclusion. Holmes is good. So is luxol fast blue. Doing both on
the same section may not be worth the effort
Addendum. Luxol fast blue isn't the best myelin stain, but it's
probably better than others if it has to follow a silver method
for axons.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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