Special Stains

Which One and Why?

Carolyn Doan
(Sakura Finetek USA)
 1. “Special Stain” Definition
“Special stain” refers to a significant number of alternative staining techniques
that are used when the H&E does not provide all the information needed to
unlock the secrets of the tissue sample in question.

Special stains are processes that generally employ a dye or chemical that has
an affinity for the particular tissue component that is to be demonstrated.

Special stains allow the presence / or absence of particular cell types,

structures and microorganisms to be viewed microscopically.

2. Mucins
Many types of stains exist for the simple purpose of demonstrating one or more
of the mucopolysaccharides found in a particular sample of tissue.

Mucopolysaccharides, or mucin, are along chains of sugar molecules found

throughout the body and are essential for life. They play an important role in cell
growth, regulating the flow of nutrients between capillaries and cells.

There are two broad categories of mucins:

 Acid mucins:
o Carry a negative charge on the mucin molecules;
o Simple;
o Complex;
o Found throughout the gastrointestinal and respiratory tract.
 Neutral mucins:
o Lack an acid group, therefore carry no charge;
o Found in the stomach epitelium, glands in the duodenum and
prostate epithelium.

Stains used to identify mucins:

 Alcian blue;
 Colloidal iron;
 Mucicarmine.

2.1. Alcian Blue

It is a basic dye containing copper, which gives it it’s blue color. The stain
molecules carry a positive charge and are attracted to negative mucins.

Adjusting the pH of the Alcian Blue allows the demonstration of subtypes of acid
mucins:
  pH 2.5 – considered a comprehensive mucin stain:
o Carboxylated mucins (carboxyl group added) = low acidity:
 Connective tissue;
 Cartilage.
o Goblet cells in Barrett’s esophagus.
 pH 1.0:
o Most sulphated mucins (sulfuric acid group added) = high acidity.

Things that normally stain positive with Alcian Blue:

 Acid mucins;
 Goblet cells;
 Mucoid capsules of organisms (cryptococcus).

Disease processes identified with a positive Alcian Blue reaction:

 Mucinous tumours:
o The term “mucinous” means that something has a lot of mucus;
o Carcinomas that are comprised of at least 60% mucus are refered
to as mucinous;
o Scientists think that the presence of mucus allows cancer cells to
spread faster. As a result, mucinous carcinomas are considered
more aggressive than regular carcinomas and are harder to
successfully treat;
o Mucinous tumors comprise about 10-15% of all adenocarcinomas
(a cancer of an epithelium that originates in glandular tissue
anywhere in the body).

Tissue Type Composition Alcian Blue pH 2.5 Alcian Blue pH 1.0

GI Tract (Glands) Neutral mucins Negative Negative
Prostate Neutral mucins Negative Negative
Epithelial cells Acid - simple Positive Negative
(Goblet)
Adenocarcinoma Acid – complex Negative Positive

2.1.1. Procedure

1) Alcian Blue (pH?) – 30 min.

2) Rinse well in running water – 2 min.
3) Counterstain in Nuclear Fast Red – 5 min.
4) Rinse briefly in running water
5) Dehydrate, clear and mount
2.1.2. Troubleshooting

 Nuclear Fast Red should be mixed well for best results;

 Care should be taken when making up the Alcian Blue to insure correct
pH of stain solution or make sure you purchase the desired pH.

2.2. Colloidal Iron