Re: Alcian blue /PAS

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From:"J. A. Kiernan" <>
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On Thu, 29 Jun 2000 Brian Fischer <> wrote:

> Inquiring minds want to know if it makes a difference to do the alcian blue 
> stain before the PAS or visa versa, Or does it not matter? 

  It does indeed make a difference - and if you can understand why, 
  it proves that a few tens of minutes in the Carbohydrate chapter 
  of any histochemistry textbook (or more learned work) was time
  well spent. The bottom line is that a mucosubstance that is
  both PAS- and alcian blue-positive will stain more strongly
  with alcian blue if the PAS reaction is done first. For a
  discussion of why this happens see Johannes ML & Klessen C (1984): 
  Alcian blue/PAS ot PAS/alcian blue? Remarks on a classical
  technique used in carbohydrate histochemistry. Histochemistry
  80: 129-132. 
    (This journal is now called Histochemistry and Cell Biology; 
     its original name was Histochemie. You can read the
     full text of this and other Springer journals on their
     web site. It's all free, without any paying, registering,
     subscribing or silly nonsense with passwords. I don't know
     if the web access goes back as far as 1982, and - sorry folks -
     also don't have the web address to hand here at home. I found 
     it easily enough with one of the regular search engines, 
     probably LookSmart.)

  More recent publications indicate that the reasons may not
  be exactly as set out by J & K in '84. However, nobody questions 
  the basic facts that:
     (a) When Schiff's reagent reacts and combines with a 
         tissue-bound aldehyde (such as periodate-oxidized
         mucus), aldehyde bisulphite compounds are formed.
         The colored Schiff-aldehyde reaction product is
         also a bisulphite addition compound.
     (b) Bisulphite addition compounds are sulphonic acids
         and, as such, are fully ionized even at very low pH.
     (c) Tissue-bound sulphonic acid anions strongly attract
         alcian blue cations. This happens at any pH including
         the usual 2.5 and 1.0 that determine the specificity
         of alcian blue staining. (It is, of course, important
         to use alcian blue at both pH 2.5 and 1.0. Reasons are
         outside the scope of this note.)
  It is not possible to make a general declaration that alcian
  blue (at either pH) should precede or follow PAS. The staining
  procedure must be tailored to the needs of the investigation.
  Simple combinations of PAS with alcian blue (pH 1 or 2.5), in 
  either order, provide only crude pictures of the macromolecular
  carbohydrates in a tissue. In association with simple chemical
  manoeuvres involving NaOH and periodic acid, the PAS method
  can be made highly specific for a wide range of sialomucins
  and even sulphated glycoproteins and proteoglycans. These
  techniques make use of reagents much less expensive than
  lectins and antibodies, and are valuable for identifying the
  level of origin of gastrointestinal tumours that contain
  mucus-producing cells. This important family of techniques
  was developed in Vancouver by the late Charles Culling and
  the late Phil Reid (both expatriate Brits) and their
  colleagues, in the 1970s-1980s. These techniques probably
  are not used as frequently as they should be, because it is
  necessary for the pathologist to understand the histochemical 
  reasoning while interpreting the appearances in the various 
  stained and control slides. (If anyone wants a reading list,
  ask & thou shalt receive. I'll post it to HistoNet if there 
  are more than two requests. This is for those with access to
  J Histochem Cytochem and Histochem J.) 

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1


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