[Histonet] Re: Phospholipids stain (Long answer)

From:John Kiernan

It depends what kind of phospholipids you
want to demonstrate, and what else you want
to do with the stained slides. 

Baker's acid haematein method allows you to 
dehydrate and make a permanent mount
because the chromium-haematein end product
is not extracted by organic solvents. The
method was shown by Adams, in the 1960s, to
stain phosphatidylcholines (lecithins) and
sphingomyelins but not other types of
phospholipid.  Bear in mind that cytomembranes
contain many kinds of phospholipid and also
other hydrophilic lipids. A histochemical
method shows mainly sites of high concentration
of membranous material such as myelin sheaths,
mitochondria and some pathological inclusions.
Baker's acid haematein method does not 
have to be a _very_ long procedure.  Baker's
original 4-day chromation (of the block, and 
then of the sections) is nowadays usually 
replaced by a 4-hour chromation of the frozen 
sections at 60C, and this is followed by staining
in acid haematein and differentiation in
borax-ferricyanide, both for 2h at 37C.
That's 8-hours for the whole procedure.
   Alternatively the chromation can take place 
overnight at 60C so the rest of the method needs 
only 4 hours (plus a few extra minutes for the 
rinses, dehydration, clearing and coverslipping).

There are plenty of other staining methods for
hydrophilic lipids (a group that includes the
phospholipids and indeed most lipids other than
fats, waxes, cholesterol and cholesterol esters).
Techniques are also available for staining 
hydrophilic and hydrophobic lipids in contrasting
colours. There are various control procedures 
(solvent extractions and chemical blocking reactions)
that can support the contention that a particular
method has really stained a certain type of lipid.

There is also an excellent, small, cheap paperback
called Lipid Histochemistry by Olga Bayliss High,
who was Adams's principal collaborator. This book
gives all the instructions, most of the explanations,
and lots of references. Do not even think of doing
lipid staining in a research context before reading
most of this book. I think it's been out of print 
for a few years. If it's not in your library, get
"Neurohistochemistry" by CWM Adams (1965). Every
library has that one; it came out in the years when
universities were spending on books, journals,
librarians, professors and scholarships for clever
students. Those were the days! Adams (1965) is a 
big book, but it contains detailed explanations that
are still valid. 

There have been several lipid histochemistry 
innovations since the 1960s, but this is not a 
rapidly changing field. It is a field in which you
need to understand the mechanisms and limitations
of the techniques. Several controls are needed
to identify a granule, droplet or smudge with even
a broad category of lipids. The information is in
the literature but anything on the internet 
(including this message from me) should be carefully 
checked against trustworthy sources. Anyone can
send a reply to a question. 

That's why you should never believe any internet 
answer until you have checked out references to
substantial sources such as articles in peer-reviewed 
scientific journals or printed books. Journals
generally have the highest standards. Book publishers
(especially for textbooks) now have really stringent 
reviewing. If you read something in a textbook
published since 2002 it was probably the "truth" 
the time of printing. Book standard were high even
in the 1960s when Adams's big fat Neurohistochemistry
book hit the market. 

If you can use thr internet to find 
Lipid Histochemistry by Olga Bayliss High,
buy it. It's a classic.

John Kiernan
London, Canada
Cyndi Bono wrote:
> I'm looking for a stain for phospholipids with similar results to the Baker's Method.  Is there a modified Bakers that has shorter timing? Or an alternative stain?
>  Cyndi Bono
> Client Services Director
> EPL, Inc.
> PO Box 474
> Herndon, VA 20172-0474
> Tel: 703-471-7060 X221
> Fax: 703-471-8447
> email: cbono@epl-inc.com
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