Re: H&E, (and how to get advice)

From:"J. A. Kiernan" <>

  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 ( 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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