Special Stains – Which One, How and Why? Part II: 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.

The Most Commonly Used Stains to Demonstrate Connective Tissue

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.

It is essential to use good Bouin's with proper incubation times for the appropriate temperature used. Complete differentiation with Phosphotungstic-phosphomolybdic acid of collagen to the point that all red is removed. If muscle red is pale, check viability of Bouin's and/or Biebrich scarlet.If collagen is too pale, decrease incubation time in 1% Glacial Acetic Acid (GLAA).

One solution stains all: muscle, collagen fibers, fibrin and erythrocytes. There are two formulations to stain collagen: blue and green.

Incubation times may vary per manufacturer guidelines.

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.

Incubation times may vary per manufacturer guidelines.

Verhoeff’s solution needs to be fresh each use due to rapid oxidation. Do not over differentiate in the ferric chloride as the dye molecules love this stuff equally, as, well as they do the tissue molecules they are bound to and will disconnect from the tissue easily. If this happens you will have to start all over. Incubate for a few seconds then rinse quickly and view microscopically looking for the elastic tissue fibers. When they are black and easily differentiated from collagen and other tissue types, stop the process.

Likewise, do not incubate too long in the van Gieson as the picric acid in solution will continue to differentiate out the bound hematoxylin.

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.

Reticulin stains use silver and rely on the argyrophilic properties of the fibers. Argyrophilic cells can adsorb silver but cannot reduce it. "Adsorb" is a surface-based process, whereas "absorb" means there is permeation through the tissue. Argyrophilic cells need a reducer/developer to convert the invisible silver salts to a visible metallic silver.

Troubleshooting the Gomori’s reticulin:

Silver solution should be made fresh with distilled water and clean glassware (acid clean is not necessary). Take care to make the silver. Using old, ammonia depleted ammonium hydroxide can cause the solution to fail. Do not use excessive ammonium hydroxide to clear the silver/sodium hydroxide solution. Excess ammonium hydroxide will cause weak staining.

How to make ammoniacal silver:

How to Demonstrate Collagen, Elastic Fibers, Reticular Fibers, Nuclei and Muscle with One Procedure

Movat Pentachrome is a super connective tissue stain. It is used to study heart tissue, blood vessels and vascular and lung diseases. Due to the excellent differentiation of collagen, elastic, fibrin and muscle fibers, the Movat can reveal subtle changes that the routine and normal special stains do not.

Incubation times and reagent sequence may vary per manufacturer, but results are similar.

Alcian blue should be used at PH 2.5. Complete removal of alkaline alcohol is essential for good staining. Verhoeff’s solution should be made fresh before each use as it oxidizes fast. Be careful not to over differentiate elastic fibers in ferric chloride. Dip once or twice then rinse in distilled water and check microscopically. Repeat as necessary. Do not use graded alcohols to dehydrate but start in absolute alcohol. Do not allow sections to dry out during the staining procedure.