On Thu, 16 Mar 2000, Carol Bobrowitz wrote:

> I am doing an Alcian Blue - PAS - Hematoxylin Stain.  I have not had alot of
> experience with Alcian Blue.  My questions are:  
> 1) Does differenciation in Acid Alcohol remove the Alcian Blue Stain?

No. Nothing removes alcian blue, because the cationic groups detach
from the molecule after it binds to the tissue, leaving behind copper
phthalocyanine, which is insoluble in pretty well everything. You can
make doubly sure of the insolubility by treating the sections with
a weak base (such as 1% ammonium hydroxide in water) for a few seconds
after staining with alcian blue. This was done in some earlier
procedures (see Pearse's Histochemistry) but not, to my knowledge,
since the early 1960s. It was probably unnecessary.

> 2) Instead of using Harris Hematoxylin should I use Mayers?

Any haemalum is OK for staining nuclei. You need to check with
a microscope and either stain for longer or differentiate, until
you have a pure nuclear stain.

Before staining a lot of slides, make sure you are doing the right
method. Alcian blue is commonly used at either pH 1 or pH 3. It also
makes a subtle but significant difference if the order is reversed, 
with the PAS done before the alcian blue. The choice of method
depends on what you want to show. Half an hour with a textbook,
so that you'll understand what's happening, would be time very
well spent. 
 
 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1