The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Alcian blue is one of the Alcian dyes introduced in 1948. Other Alcian dyes include Alcian green and Alcian yellow.
Alcian Blue stains much more slowly than other basic dyes, and differential staining by the dye is greatly influenced by differences in the rate of staining of various basophilic components. Epithelial mucin stains most rapidly, followed by mast cell granules, pericapsular cartilage matrix, interstitial cartilage matrix, nuclei and cytoplasmic chromidial substance (presumably RNA), in that order.
Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes. This is a family of polyvalent basic dyes that contains 2-4 positively charged isothiouronium groups. Alcian blue is a phthalocyanine dye that shows specificity for substances such as glycosaminoglycans and acid mucins.
Alcian blue causes Acid mucins and mucosubstances to turn blue and the nuclei to appear reddish pink when using counterstain neutral red for the substrate. Alcian blue dyes are water soluble and they appear blue because of the copper content that they have. They also attach to sulfate and carboxylated acid mucopolysaccharides and glycoproteins and dye binding is purely electrostatic.
The pH of the alcian blue staining solution is a critical factor in the staining procedure and has a direct effect on the category of mucins that can be demonstrated with the procedure. The pH of alcian blue staining solutions should range from 0.4 to 2.5, with a pH of 1.0 and 2.5 being used most commonly.
This staining is performed to look for mucoid degeneration and to identify acid mucins which are released by various connective and epithelial tissue tumors.
The aggregation of Alcian Blue in solution, as assessed by changes in the absorption spectrum, is markedly affected by dye concentration, dyebath temperature, additives such as salt or urea, and also by the age of the solution, the dye being notably unstable under some conditions.
- The section is hydrated with distilled water
- It is submerged in alcian blue (time in the alcian blue can vary)
- It is then washed in tap water, and rinsed in distilled water
- Nuclear-fast red is added as a counterstain for 5 minutes
- It is then dehydrated with ethanol and cleared using xylene
- The section is mounted using a resinous medium