Re: H&E, (and how to get advice)
|From:||"J. A. Kiernan" <firstname.lastname@example.org>|
Dear "Circle" (whoever you are),
Here is an incomplete answer, with no real authority, to one
or two of your questions sent to HistoNet. To get better
answers you will have to explain your problem in more detail.
On Sun, 5 Nov 2000, Circle :> wrote:
> I learnt from histology books that H&E is usually used in
> staining. However, the colour is sometimes different. Is H&E applicable in
> most staining? How can I use it to distinguish vascualr smooth muscle
> cells and endothelial cells?
In a normal blood vessel there should be no difficulty, because
the endothelial cells form a monolayer lining the vessel. In
pathological or experimental material you may well have difficulty.
All the H & E method does is stain nuclei blue and then stain
everything red (which usually makes the nuclei look purple). In
practice the eosin counterstain is largely restrained to pink
rather than red, and with skill and care it is possible to attain
various shades of pink in collagen and different types of cytoplasm,
including an almost orange colour in erythrocytes.
H & E is popular because pathologists (who rarely do the staining
themselves) have for many years considered it the "routine" or
"standard" technique. There are plenty of other staining methods
that are easier to do, do not require microscopic control to get
the best results, use cheaper chemicals, and provide more informative
colour schemes in the sections. Research workers (who often have to
do their own staining and teach the art to graduate students and new,
untrained technicians) often prefer methods that are both technically
simpler and more informative than H & E.
However, even a simple method that gives you 3 or more colours will
not necessarily provide certain identification of endothelial cells,
smooth muscle cells, fibrocytes and other cells in an abnormal vessel
or in a mixture of cultured cells. You need to use immunocytochemistry,
with proper positive and negative controls, to do the job with
reasonable certainty. Antibodies to Factor VIII (the classical
anti-haemophilic globulin) are frequently used as endothelial markers,
and antibodies to actin label smooth muscle (though not with absolute
specificity; there are different sorts of actin, and probably you
will get a reply with more detailed advice from a Histonet subscriber
with expertise in that field).
Your use of a pseudonym ("Circle") may offend some Histonetters, and
could reduce the number of helpful replies. Just about everyone who
asks or answers a question reveals his or her true name, and for the
most part we are all on first-name terms even though many (probably
most) of us have never met one another in person.
From your questions it is clear that you are a beginner in the field
of histotechnology. As a fairly frequent contributor I would like to
say that questions from you and from other beginners are VERY WELCOME,
and you should not feel ashamed to ask questions about simple things
that you could look up in a histotechnology textbook. There are lots
of enquiries just like yours. Often the response (from multiple
Histonetters) is "Go and look it up in the library," followed by
advice about which books or journals to look for. You may also be
referred to the HistoNet archives, a web site where you can search
for earlier Histonet exchanges (www.histosearch.com). Remember that
references to papers in peer-reviewed scientific journals have more
authority than books and much more authority than statements that
do not include any references (such as this Histonet reply).
Keep in touch with HistoNet, and consider showing yourself as more
than an array of an infinite number of coalescent points, at the
same distance from another point (the centre) in the same plane.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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