Alcian Blue (2.5) Staining Procedure

Purpose:
This standard operating procedure (SOP) establishes the standard for staining polysaccharides and mucosubstances using the Alcian Blue staining procedure.

Reagents and Materials:

Reagents

3% Acetic Acid
Acetic Acid 3.0ml
Distilled water 97.0ml

1% Alcian Blue Ph 2.5
Commercially prepared by Newcomer Supply Co.
Expiration date provided by manufacturer.
Store at room temperature.

Nuclear Fast Red
Commercially prepared by Newcomer Supply Co.
(catalog #12552)
Expiration date provided by manufacturer.
Store at room temperature.

Materials
Tissue slides and control slides for special stain requested.
Microwave to bake slides.
Staining rack, 20 slide capacity.
Coverslips and synthetic mounting media.

Hazards and Precautions:
See Material Safety Data Sheets.

Sample Preparations:
Paraffin tissue section slides cut at 4 microns.

Calibration and Standardization:
A tissue section slide containing polysaccharides and mucosubstances is stained with each procedure.

Procedure:

1. Deparaffinize and hydrate to distilled water.

2. 3% Acetic Acid – 3 minutes.

3. Place in 1% Alcian Blue Ph 2.5 in a plastic coplin jar with lid loosely applied. Microwave at 70% power for 30 seconds.

4. Distilled water rinse.

5. Nuclear Fast Red – 5 minutes.

6. Distilled water rinse.

7. Dehydrate, clear and coverslip.

Positive control: intestine or lung with goblet cells

Results:
All polysaccharides and mucosubstances – blue
Sulfomucins – blue
Sialomucins – red
Background – pink

Note: PAS counterstain may be used:
Beginning at step 5, perform PAS technique up to sulfurous rinse.

Figure 1. Canine chronic bronchitis. H&E left; alcian blue pH 2.5 – PAS right: sulfomucins and sialomucins stain blue.

Effective: July 1, 2004
Cherie Chapman
James R. Turk

References:

Armed Forces Institute of Pathology Laboratory Methods in Histotechnology, eds. Prophet EB, Mills B, Arrington JB, Sobin LH, American Registry of pathology, Washington D.C. , 1992, pp164.

Sheehan DC, Hrapchak BR: Theory and Practice of Histotechnology, 2nd Ed, CV Mosby, St. Louis, 1980, pp. 172. Wheeldon EB, Pirie HM, Breeze RG. A histochemical study of the tracheobronchial epithelial mucosubstances in normal dogs and dogs with chronic bronchitis. Folia Vet Lat. 1976 Jan-Mar;6(1):45-58.

Tintut Y, Alfonso Z, Saini T, Radcliff K, Watson K, Bostrom K, Demer LL. Multilineage potential of cells from the artery wall. Circulation. 2003 Nov 18;108(20):2505-10.

Wheeldon EB, Breeze RG, Pirie HM. Animal model: chronic bronchitis in dogs. Am J Pathol. 1979 Jul;96(1):355-8

Turk JR, Rantanen NR: Chronic bronchitis, cardiomegaly and medial hypertrophy of small pulmonary arteries in a dog. J Small Anim Practice 23:719 723,1982.

Sarbia M, Donner A, Franke C, Gabbert HE. Distinction between intestinal metaplasia in the cardia and in Barrett's esophagus: the role of histology and immunohistochemistry. Hum Pathol. 2004 Mar;35(3):371-6.