Fixation on Histology

The Alcian Blue Stain for Histology


Like many of the other dyes currently used in histology, Alcian blue started out in the textile industry, originally manufactured in the UK in the 1940’s by Imperial Chemical Industries. Testing of the dye for biological purposes began in the 1950’s, ultimately resulting in the Alcian Blue 8GX used today.

Alcian Blue is a stain that is used to visualize acidic epithelial and connective tissue mucins. Mucins are a type of carbohydrate and are found in the GI tract and respiratory tract. Acidic mucins have a negative charge. The alcian blue dye molecule has a copper-containing pthalocyanine ring linked to four isothiouronium groups which have a positive charge. The dye molecule binds with the sulphur and/or carboxyl groups of the mucin depending on the pH used. The pH will determine which mucins are stained. With a 3% acetic acid solution with pH 2.5 it will stain both sulphated and carboxylated acid mucopolysaccharides and glycoproteins. At pH 1.0 it will only stain sulphated acid mucopolysaccharides, so the more highly acidic mucins. The blue color of the stain is produced by the copper in the dye molecule. pH 2.5 alcian blue is used to diagnose Barrett’s esophagus, as it will also stain goblet cells. It is also used in mesothelioma, atherosclerosis and adenocarcinomas.

Alcian blue will color the acidic mucins blue but will not color neutral mucins. If you need to visualize the neutral mucins as well, alcian blue can be followed with PAS, Periodic Acid Schiff, which stains the neutral mucins magenta. Cells that have both acidic and neutral mucins may show up dark blue or purple. PAS, unlike alcian blue, doesn’t rely on looking for the acidic groups, but instead the structure of the saccharides. In this stain, the periodic acid oxides glycogen to form aldehyde groups which then bind with the Schiff’s reagent.

Alcian blue is not the only stain for mucins. Mucicarmine is another stain traditionally used for mucins, however it can only be used for epithelial tissue, not both epithelial and connective tissue, as alcian blue can. Mucicarmine was used primarily for differentiation between non mucin producing squamous cell carcinomas and mucin positive adenocarcinomas, but is not done as often now as IHC has largely replaced it.

To learn more about alcian blue, and other stains on the HT ASCP exam, check out NSH’s HT Prep Course, available on

Alcian Blue
Alcian Blue Stain

Alcian Blue PAS
Alcian Blue PAS Stain