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Special Stain - ABPAS and Alcian Blue

  • Mucin staining
  • ABPAS technique
  • Alcian Blue
  • Mucins
  • Acid Mucins
  • Type acid mucins

Alcian Blue

This technique is used to stain the mucins in tissues. As shown below, the alcian blue reacts with mucins to stain them blue. The mucin reaction is pH dependant; with the normal pH used for staining is pH 2.5 which stains up acid mucins.

ABPAS - Alcian Blue Periodic acid schiff

This technique can be combined with the PAS to differentiate between acidic and neutral mucins, with the alcian blue staining up the acidic mucins, while PAS stains the neutral mucins. This is valuable as it allows differentiation of the mucin types. It is also important to stain in the correct order, as staining the PAS before the Alcian blue can lead to problems with the staining.

ABPAS technique

Below is how to perform the ABPAS technique
  1. Take sections to water
  2. Stain in alcian blue solution for 5 minutes.
  3. Wash in water.
  4. Treat with periodic acid for 10 minutes.
  5. Wash in water.
  6. Treat with Schiffs Reagent for 15 minutes at room temperature.
  7. Wash in running hot water for 5 minutes.
  8. Dehydrate, clear and mount

Results

Acid Mucins

Blue

Neutral Mucins

Magenta


Photo – x10 Alcian Blue PAS staining

Alcian Blue

This technique is when the Alcian Blue is carried out on its own.

  1. Take sections to water
  2. Stain in Alcian Blue solution in a coplin jar for 5 minutes
  3. Wash in water
  4. Stain in Nuclear Fast Red solution for 5 minutes
  5. Wash in water
  6. Dehydrate, clear and mount
  7. Results

    pH2.5 Acid mucins

    Blue

    pH1 Amyloid

    Green

    Nuclei

    Red

    Photo - Alcian Blue in colon x10

The two main types of mucins found in the body are listed in the table below.

Main mucin types and locations in the body

Mucin Type

Structure and Location within the body

Acid Mucins

Positively charged and form electrostatic bonds with tissue polyanions bearing either carboxyl or sulphate groups. These are mostly found in the colon.

Neutral Mucins

Found in the gastric lining cells in the stomach and in brunner glands of the duodenum. Also present in the alimentary and respiratory tract goblet cells and prostate glands.

The main difference between the different types of mucins is that the neutral mucins contain a high content of neutral monosaccharides like mannose, galactosamine and galactose. While the more acidic mucins contain a carboxyl group and also an N-acetyl group which gives it the acidic pH. The locations of each of the mucin types are important as they can be used in diagnosis, one example is when determining the origin in the body of an adenocarcinoma based on what mucin type is being expressed.

The acid mucins subtypes are listed in the table below.

Acid mucin sub division and location

Acid mucin sub division

Location in the body

Strongly sulphanated

Found in connective tissue and epithelial, these can be found in skin, cartilage, bone, blood vessels and umbilical cord. They will react at highly acidic pH 0.5.

Weakly suphanated

Epithelial tissue, normally found in colon goblet cells. They stain up with cationic dyes at pH 1

Carboxylated - sialomucin

There are two forms one is an enzyme labile which are digested by the enzyme sialidase, commonly found in bronchial, submucus glands, submandibular salivary glands and goblet cells in the small intestine. While the enzyme resistant form are epithelial in origin and are found in colon goblet cells.

Hyaluronic Acid

This is found in connective tissue in the umbilical cord and is also an important constituent of the synovial fluid. This has a reactive site on the carboxyl group and stains up at pH 2.5.

Sulphanated Sialomucins

These react with sulphanated mucins and are extracted by the enzyme sialidase and have been found in tumours of the prostate