I.

CONNECTIVE TISSUE
Connective tissue supports the body by providing a matrix that connects and binds the cells and
organs. There are three types of connective tissue in the body.
Collagen is a strong protein and is a main component of ligaments and tendon. It is also responsible
for skin elasticity and is the most visible type which can be visualized with an H&E.

Elastic fibers are located in the skin and walls of blood vessels. They are composed of elastin - a
protein that is flexible and allows many tissues in the body to resume their shape after stretching or
contracting and are not visualized by an H&E.
Reticular fibers are composed of collagen and form a delicate framework around nerve fibers, fat
cells, lymph nodes, and smooth and skeletal muscle fibers. They are also not visualized by an H&E.

1. Trichrome - Masson
Three dyes are used to selectively stain including muscle (red), collagen fibers (blue) erythrocytes
(red) and nuclei (black).
The dyes are used to stain liver biopsies to determine the degree of fibrosis as in cirrhosis as well
as kidney biopsies to accentuate the basement membrane and demonstrate immune deposits. The
dyes are also used to differentiate between smooth muscle and collagen in tumors and verify
increase of collagen.

The Process
The Protocol – Incubation times may vary per manufacturer guidelines
1. Bring slides to water
2. Incubate in Bouin's – acts as a mordant to bind the dye to the tissue.
• Room temperature Usually overnight
• 56c to 60c 10-15 mins
Note: If heating Bouin's in microwave, do not heat with slides in
solution; heat Bouin's, remove from microwave, place slides in
Coplin jar, seal with lid and incubate outside microwave.
3. Rinse well in running water to remove all traces of Bouin's
4. Weigert's hematoxylin 5-10 mins
5. Rinse in running water 5 mins
6. Biebrich scarlet 15 mins
7. Rinse in running water
8. Phosphotungstic-phosphomolybdic acid 15 mins
9. Straight into aniline blue (no rinse) 5 mins
10. Straight into 1% glacial acetic acid 1-3 mins
11. Rinse in running water
12. Dehydrate, clear and mount

The Results
• Muscle - red
• Collagen - blue
• Nuclei - black or blue
• Fibrin - red
2. One Step Trichrome - Gomori's
One solution stains all: muscle, collagen fibers, fibrin and erythrocytes. There are two formulations
to stain collagen: blue and green.

The Process

The Protocol
Incubation times may vary per manufacturer guidelines.
1. Bring slides to water
2. Incubate in Bouin's – acts as mordant to bind the dye to the tissue
• Room temperature usually overnight
• 56c to 60c
Note: If heating Bouin's in microwave, do not heat with slides in
10-15 mins
solution; heat Bouin's, remove from microwave, place slides in
Coplin jar, seal with lid and incubate.
3. Rinse well in running water to remove all traces of Bouin's
4. Weigert's hematoxylin 5-10 mins
5. Rinse in running water 5 mins
6. One step blue/green solution 15 mins
7. Straight to 1% glacial acetic acid 1 min
8. Rinse in running water
9. Dehydrate, clear and mount

The Results
3. Verhoeff-van Gieson's Elastic Stain
Verhoeff-van Gieson's Elastic Stain is used to identify the atrophy of elastic tissue as in emphysema,
evidence of vascular diseases (arteriosclerosis), and the invasion of tumors into vessels.

The Process

The Protocol
Incubation times may vary per manufacturer guidelines.
1. Bring slides to water
2. Verhoeff elastic stain-working solution (per manufacturer) 15 min
3. Rinse well with running water
4. Differentiate in 2% ferric chloride 1-2 mins
5. Rinse in running water 5-10 mins
6. 5% sodium thiosulfate 1 min
7. Rinse in running water
8. Counterstain in van Gieson's solution 30 sec -1min
9. Skip 95% and quickly dehydrate in 100 x 2
10. Clear and mount

The Results
Elastic fibres: black
Nuclei: black
Collagen: red
Other tissues: yellow
4. Gomori's Reticulin Fiber
Reticulin fibers support the body and are common in the liver, spleen and kidneys. Characteristic
reticulin patterns can help diagnose cirrhosis of the liver, early fibrosis in bone marrow, tumors
including: hemangiosarcomas - tumor of cells that line blood vessels, fibroblastic tumors, and
rhabdomyosarcomas - tumor of muscles that are attached to bone. They can also help diagnose
epithelial versus non-epithelial tumors.

The Process

The Protocol
1. Bring slides to water
2. Oxidize in potassium permanganate 10 min
3. Rinse well in running water 3 mins
4. Oxalic acid 1 mins
5. Rinse well in running water 2 mins
6. Sensitize in ferric ammonium sulfate 15 mins
7. Rinse in distilled water 2 mins
8. Impregnate in ammoniacal silver 2 mins
9. Rinse in distilled water 1 min
10. Develop in 20% unbuffered formalin 1 min
11. Rinse well in running water 2 mins
12. Tone in 0.1% gold chloride 3-5 mins
13. Rinse in water 1 min
14. 5% sodium thiosulfate 1 min
15. Rinse in water 1 min
16. Counterstain in nuclear fast red 3-5 mins
17. Rinse briefly in water 15 sec
18. Dehydrate, clear and mount

The Results

5. Weigert’s Resorcin-Fuchsin Stain

This is a combination of stains used in identifying elastic fibers. Often, orcein or a combination of
resorcinol and fuchsin are used for staining. For counterstaining cell nuclei, nuclear fast red or
hematoxylin is also used. After applying, elastic fibers appear blue to black colored while cell nuclei
appears red or blue.
The complex formed from the basic fuchsin, an iron resorcin lake, binds to the elastic fibers,
resulting in the blue-black staining. The nuclear detail is stained with an iron hematoxylin which will
not over differentiate in the acidic elastic stain solution.
 II. CARBOHYDRATES
DYES FOR MUCINS
Mucins are part of a complex group called carbohydrates. They can be simply defined as sugars and
starches. Carbohydrates are divided into monosaccharides, polysaccharides and
mucopolysaccharides.

Mucins are mucopolysaccharides which are explained as long chains of sugar molecules found
throughout the body and essential for life and significant in maintaining the structural integrity of
bone, cartilage, skin, elastic tissue and membranes

There are two broad categories of mucins:

a. Acid mucins which carry a negative charge on the mucin molecules and can be classified
as either simple (carboxyl group added) or complex (sulfuric acid group added). They are found
widely throughout the gastrointestinal and respiratory tracts.
b. Neutral mucins which lack acid groups and carry no charge. They are found in the
epithelium of the stomach and the Brunner’s glands of the duodenum. They protect the lining of the
duodenum from stomach acid content, help enable absorption by providing an alkaline environment
for digestive enzymes, and lubricate intestinal walls and prostate epithelium.

A. Dyes for Acid Mucins

1. Alcian Blue
Alcian Blue is a basic dye containing copper, which gives it the blue color. The stain molecules carry
a positive charge and are attracted to the negative mucins. Adjusting the pH of the Alcian Blue
solution allows the demonstration of sub-types of acid mucins.

pH 2.5 – considered a comprehensive mucin stain will demonstrate:

• Carboxylated (low acidity) simple mucins such as connective tissue and cartilage
• Goblet cells in Barrett’s esophagus

pH 1.0 will demonstrate sulphated (high acidity) complex mucins in:

• Large intestinal goblet cells

• Bronchial serous glands
• Adenocarcinomas

Note: Alcian Blue does not stain neutral mucins

The Process
The Protocol – Incubation times may vary per manufacturer
1. Run slide to water
2. Alcian Blue – careful to select appropriate PH 30 min
3. Rinse well in running water 2 min
4. Counter stain in nuclear fast red 2 min
5. Rinse briefly in running water
6. Dehydrate, clear, and mount

The Result

2. Colloidal Iron
Colloidal Iron is used to distinguish acid mucins. It is often used to replace the Alcian Blue stain due
to its greater sensitivity for acid mucins with detection of very small quantities. It is used for
diagnosing some renal cell carcinomas and some mesotheliomas – a rare form of cancer that
develops from transformed cells originating in the mesothelium (protective lining that covers many
internal organs).

The Process
Positively charged iron ions (ferric) are attracted to the negative mucin molecules. The attached iron
ions are then treated with potassium ferrocyanide/HCL to form visible bright blue deposits.
The Protocol – Incubation times may vary per manufacturer
1. Run slides to water
2. Incubate in 12% GLAA (glacial acetic acid) 12 min
3. Straight to working colloidal iron solution 60 min
4. Rinse in 12% GLAA 3 min
5. Rinse in second 12% GLAA 3 min
6. Ferrocyanide – Hydrochloric acid solution 20 min
7. Rinse in running water 2 min
8. Counterstain in nuclear fast red 5 min
9. Rinse briefly in running water
10. Dehydrate, clear, and mount

The Result

2. Mucicarmine
A very specific stain for mucin of epithelial origin
Mucin is a secretion produced by a variety of epithelial cells and
connective tissue cells. In certain intestinal inflammations or
carcinomas, an excess of mucin is secreted by the epithelial
cells.

Mucicarmine staining is also useful for staining encapsulated

fungi like cryptococcus.

The Mucicarmine stain is also used to differentiate between

mucin negative squamous cell carcinoma and mucin positive
adenocarcinoma.
The Process
Carmine, a dye obtained from the pulverized bodies of the cochineal beetle, is bound into solution
with aluminum salts (a process called mordanting) much the same as hematoxylin. This produces a
positively charged complex which is attracted to the negative acid mucin molecule.

The principle of mucicarmine staining is based on the presence of aluminum that forms a chelating
complex with carmine. This changes the charge of the carmine molecule to a positive charge which
allows it to bind to low density acidic substrates such as mucins.

The Protocol – Incubation times may vary per manufacturer

1 Run slides to water
2 Working Wiegert’s hematoxylin 5-10 min
3 Rinse in running water
4 Working mucicarmine solution 20-30 min
5 Rinse in running water
6 Metanil yellow 1-3 min
7 Rinse briefly in running water
8 Dehydrate, clear and mount

B. Dyes for Neutral Mucins

Periodic acid Schiff - PAS
Periodic acid - Schiff is the most versatile of the carbohydrate stains demonstrating neutral mucins
and as noted below, glycogen. It recognizes non-sulfated, simple acid mucins in epithelial cells and
does not rely on the negative charge of the mucin cells but the structure of the saccharide units.
The Process

The Protocol – Incubation times will vary per manufacturer

1 Run slides to water
2 Oxidize in 0.5% periodic acid 5 min
3 Schiff’s solution 15 min
4 Rinse in running water 5 min
5 Mayer’s hematoxylin 1 min
6 Rinse in running water 1 min
7 Dehydrate, clear, and mount

DYES FOR AMYLOID

Amyloid is an intercellular material that is deposited in various tissues such as heart, muscle,
skin, liver, spleen, kidneys and brain. It was originally classified as a carbohydrate, but
amyloid is now classified based on the structure of its fibril subunit proteins.

Congo Red
Congo red is a dye with a selective affinity for amyloid. Amyloid deposits are formed in tissues
spontaneously or in association with a wide range of disease conditions. The deposits are
intercellular and may become large enough to cause damage to surrounding tissues. Depending on
the type of underlying disease, amyloid deposits may form in muscle, heart, skin, tongue, liver,
spleen, kidneys or adrenals.

The Result
Amyloid - pink to red (apple-green with polarized
light)
DYES FOR GLYCOGEN
Glycogen is a simple polysaccharide that is widely distributed throughout the body. It is
found in greatest amounts in the liver, hair follicles, endometrial glands, vaginal and
ectocervical epithelium, and cardiac and skeletal muscles.

How to demonstrate glycogen:

Periodic acid Schiff's – PAS are used to demonstrate glycogen in the liver, the basement membrane,
certain tumors of the bladder, kidney, pancreas and ovary, and fungus.

The presence of glycogen and its distribution patterns are significant in diseases such as:
• Glycogen storage disease of the liver
• Pompe disease - the build-up of glycogen causes progressive muscle weakness (myopathy)
throughout the body and affects various body tissues, particularly in the heart, skeletal
muscles, liver and nervous system
• Rhabdomyosarcoma – a connective tissue cancer
• Mesothelioma

The Process

Results:

Slide A shows a strong PAS reaction Slide B staining disappears after diastase digestion
Areas that are PAS positive in the untreated slide A, and PAS negative in the treated slide B, are
assumed to be true glycogen. PAS positive in both is other PAS positive material not broken down
by the enzyme.
Diastase is a malt extract containing alpha and beta amylase extracts which is an enzyme
that aids in the conversion of starches and sugars. This amylase breaks down glycogen into
smaller sugar molecules which are then washed out.