Alcian Blue

Purpose:

• To demonstrate the presence of various acidic mucins in tissue. The Alcian Blue should never stain neutral mucins. (Alcian Blue)

Control Tissues:

• Small Intestine, Appendix, Colon (Alcian Blue Stain Kit (PH 2.5, Mucin Stain) (Ab150662) | Abcam)

Staining Pattern:

• Alcian Blue 2.5- All Acid Mucins will stain bright blue. These include sulfomucins, sialimucins, proteoglycans
• Alcian Blue 1.0- The most acidic mucins will stain bright blue.

Biochemistry Theory:

• Alcian Blue 2.5 demonstrates acid mucopolysaccharides. A 3% acetic acid solution is added to the Alcian Blue solution to bring the pH to 2.5. At a pH of 2.5 both carboxylated and sulfated mucopolysaccharides and glycoproteins will be stained. (Carson, 2001)
• Alcian Blue 1.0 demonstrates sulfated muco-substances. A 0.1N hydrochloric acid is added to the Alcian Blue solution. This brings the pH down to a low 1.0 pH. At this pH only sulfated acid mucopolysaccharides and glycoproteins will be stained. (Carson, 2001)
• Alcian Blue with Hyaluronidase- there is a digestion of  muco-substances that contain hyaluronic acid, chondroitin sulfates A and C. The digestive agent is testicular hyaluronidase After this digestion only the epithelial and connective acid muco-substances are present and stained. (Carson, 2001)
• Alcian Blue with PAS-This stain will demonstrate neutral and acidic muco-substances as different colors. A 2.5 pH Alcian Blue solution stains the acid muco-substances blue, then a PAS solution stains the neutral polysaccharides purple (Carson, 2001)

• Alcian Blue is a copper phyhalocyanin (refers to chemical structure) basic dye.  The copper gives it the classic bright blue hue. It is theorized that a salt linkage is formed when Alcian Blue solutions come into contact with the appropriate muco-substances. (Carson, 2001)

• The strength of the bonds that are created with Alcian Blue in the tissue are thought to be correlated to the deepness of the blue dye. This meaning that the stronger the blue color observed on the tissue- the stronger the salt linkage is. To create more robust staining that will survive other downstream staining efforts, one should increase the incubation time of the Alcian Blue. (Disbrey & Rack, 1970)

Images:

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Sample Protocol: