Re: Alcian Blue w/ & w/o Hyaluronidase

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From:"J. A. Kiernan" <> (by way of histonet)
To:histonet <>
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On Fri, 26 Feb 1999 FRUGE.JOSEPH@TUCSON.VA.GOV wrote:

> We have consistently had difficulty with the Alcian Blue with and without
> Hyaluronidase Stain.  We are using a section of cartilege as our positive
> control. We use a Phosphate buffer solution at pH 5.6 with testicular
> hyaluronidase purchased from Sigma Chemical.  We use 25 mg of
> hyaluronidase to 50 cc of buffer. We are using a 3% solution of Alcian
> Blue ph 2.5.  Any help out there?

 Based on principles, experience with alcian blue, but not experience
 of trying to stain specifically for hyaluronic acid: At pH 2.5 alcian
 blue stains all polyanions except nucleic acids. At pH 1, alcian blue
 (or any other basic dye) stains only those polyanions that owe their
 acidity to half-sulphate ester groups: the chondroitin sulphates,
 heparin and a few others. Cartilage matrix contains lots of chondroitin
 sulphates, so it stains with alcian blue and all other basic dyes at
 any pH, however acid. Hyaluronic acid is a polycarboxylic acid present
 in many tissues. It would never stain with alcian blue at pH 1 but it
 might well stain at pH 2.5.

 In order to get a chance of staining hyaluronic acid with a basic dye
 like alcian blue you must first permanently get rid of all the half-
 sulphate esters of chondroitin. This is done by METHYLATION: 1 ml of
 conc. hydrochloric acid in 100 ml methanol for 48 hrs in an airtight
 container at 55-60C. Methylation also esterifies the carboxyl groups
 of hyaluronic acid, but these can be restored by SAPONIFICATION: about
 20 m in 0.5% KOH in 70% ethanol. In a section that has been methylated
 and then saponified, extracellular staining by alcian blue at pH 2.5
 is almost certainly attributable to hyaluronic acid. A digestion
 with hyaluronidase (done before any of the other procedures) will
 prove the point. The connective tissue of the umbilical cord, known
 as Wharton's jelly, is a traditional example of material rich in
 hyaluronic acid.

 Regarding hyaluronidases, these are not all equal. See Pearse's
 Histochemistry (4th ed Vol 2, 1985: Chapters 13 and 15-pt I) for
 discussion of specificity, caution in interpreting results etc.

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

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