Have cut and pasted our method from our Staining Porcedures Manual 
for your reference.





Department of Anatomical Pathology
Royal Hobart Hospital

ALCIAN BLUE / HAEMATOXYLIN AND EOSIN STAIN
FOR ACIDIC MUCINS

SAFETY PRECAUTIONS:
 CAUTION: This department's policy is to treat all dyes as potential
 carcinogens. Use a fumehood and protective clothing when handling
 hazardous chemicals. A laboratory gown, gloves, safety glasses and
 dust mask is a minimum standard.

PRINCIPLE:
 Alcian blue is a copper phthalocyanin basic dye. At pH2.5
 alcian blue stains both sulphated and carboxylated acid 
mucopolysaccharides and sialomucins (glycoproteins). Alcian blue can 
be used to separate and thus identify the different acid mucins by 
using alcian blue at varying pH. Sulphate esters react at a lower pH 
than do the carboxyl groups.
 SECTION:         3u paraffin section
CONTROL SECTION:  Small intestine
REAGENTS:
 1.  Celestine blue-See method H1 in this manual for reagent
         preparation.
  2.  Harris haematoxylin-See method H1 in this manual for
       reagent preparation.
  3.  Eosin-See method H1 in this manual for reagent
         preparation.
  4.  Alcian blue ( pH 2.5 and / or pH 1.0 ).   Filter before use.
      ( pH2.5 -  1%Alcian Blue in 3% acetic acid. pH 1.0 - 
1%alcian blue in 0.1MHCL
METHOD: 
1.  Dewax sections in xylene and hydrate through graded       
 alcohols to water.
 2.  Stain in  celestine blue  --- 1 min 30 sec
 3. Wash well in tap water.
 4.  Stain in Harris haematoxylin --- 10min.
 5.  Wash in tap water. 
6.  Differentiate in 1% HCl in 70% alcohol --- 4 sec 
7.  Wash in tapwater.
 8.  Blue in freshly ammoniated tap water ( 8 drops / 500 ml ) --- 30 
 sec
 9.  Wash in tap water, rinse in distilled water.
10. Stain in alcian blue solution --- 5 min. 
11. Wash in tap water.
 12. Stain in eosin --- 2 min.
 13. Rinse in tap water.
 14. Dehydrate through graded alcohols,clear in xylene and mount  
      with a resinous mountant.
RESULTS:
  At pH 2.5 most acid mucins --- blue
 (except some of the strongly sulphated groups)
 At pH 1.0 only weakly and strongly sulphated acid mucins --- blue
REFERENCES:
 Bancroft J.D. Stevens A. (1990), Theory and Practice of Histological
 Techniques, Churchill Livingstone, 3rd Edn. pp. 193.




Issue Date: 20/09/1999	Amendment Date:	Review Date:
Authorised By: RD Rainbow / Tony Van Galen	Amended By:	Reviewed By:
Routine Staining Method Manual								Page 1 of 1



Hope this helps
Ron Rainbow
Scientist in Charge
Department of Anatomical Pathology
Royal Hobart Hospital
GPO Box 1061L
Hobart
Tasmania
Australia 7001
Ph 0362228771
Facsimile 0362228191
eMAIL:  ron.rainbow@dchs.tas.gov.au