Re: [Histonet] alcian blue/pas

From:John Kiernan

Dear Patsy Ruegg,

If you're working with frozen sections of a tissue that contains
 beta-galactosidase, the usual method for staining the lac-z gene
product should not give a false-positive because the pH optima
for the animal (lysosomal) and E. coli galactosidases are widely
separated. I'm assumong that you're detecting the marker enzyme
with the usual indogogenic method, using the substrate commonly
called X-gal. (I can't be bothered to type the real name.)

That said, the product of the indigogenic X-gal reaction has a
rather unpleasing greenish blue colour. Material stained with
alcian blue has a turquoise colour - a lovely balance of blue and
green. The X-gal product and alcian blue colours are not the
same, but they are similar. 

There are indigogenic beta-galactosides that yield green and red
indigoid dyes, but I have found no published reports of their use
with tissue sections and microscopes. Please let me know if I've
missed something in this field. The insoluble indigoid pigment
formed from X-gal is formed more quickly than the ones from other
indoxyl beta-galactosides. The rather dirty-looking blue-green
product is deposited near the molecules of the galactosidase.
"Near" in this context means in the cytoplasm rather than the
nucleus, and perhaps at the basal or apical end of an epithelial
cell. This is LM, not EM enzyme histochemistry.

The X-gal method is "standard" among molecular biologists. You
may want to consider alternatives to an alcian blue & PAS
counterstain.  The bromo-chloro-indigo product of the X-gal
reaction is almost as indestructible as copper phthalocyanine
(the product of alcian blue staining). There are several alcian
blue-PAS and PAS-alcian blue methods (the sequence affects the
result, and in a logical way). 

What is expected of the counterstain to your lac-z reporter gene?
If greeny-blue is mandatory for lac-z localization, all the other
colours are up for grabs. 

John Kiernan
Anatomy, UWO
London, Canada.
Patsy Ruegg wrote:
> Can someone steer me to a good alcian blue/pas stain for frozen sections.
> We have mouse tissue labeled with Lac Z and someone mentioned that cells
> making mucous could actually non-specifically stain with the blue Lac Z, so
> we need to prove the positive stain we have with Lac Z is real and not
> because the cells are making mucous.
> Patsy
> Patsy Ruegg, HT(ASCP)QIHC
> IHCtech, LLC
> Fitzsimmons BioScience Park
> 12635 Montview Blvd. Suite 216
> Aurora, CO 80010
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