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

Define "Special Stain"

"Special stain" is a term used to refer to many alternative staining techniques that are used when the traditional H&E does not provide all the information the pathologist or researcher needs from a tissue slide. "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. They allow the presence/or absence of certain cell types, structures and/or microorganisms to be viewed microscopically. "Special stains" are not the same as and existed long before immunohistochemical (IHC) and/or molecular techniques (probes).

Connective Tissue - Introduction

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.

Collagen – elastic and reticular fibres

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

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 Process – Trichrome - Masson's

The Results

Kidney showing immune deposits
Kidney showing immune deposits

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 minutes
Note: if heating Bouin’s in microwave, do not heat with slides in solution; heat Bouin’s, remove from mv, place slides in coplin jar, seal with lid and incubate outside MV.
3. Rinse well in running water to remove all traces of Bouin’s
4. Weigert’s hematoxylin 5-10 min
5. Rinse in running water 5 min
6. Biebrich scarlet 15 min
7. Rinse in running water
8. Phosphotungstic/phosphomolybdic acid 15 min
9. Straight to analine blue (no rinse) 5 min
10. Straight to 1% glacial acetic acid 1-3 min
11. Rinse in running water
12. Dehydrate, clear and mount

 

Troubleshooting the Trichrome:

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% GLAA. Nuclei - black

One Step Trichrome - Gomori's

One solution stains all: muscle, collagen fibers, fibrin and erythrocytes. There are two formulations available: blue - for those who prefer the collagen blue as in Masson's, green - for those who prefer the collagen green, and all other results are similar to Masson's.

The Process

Verhoeff's van Gieson Elastic Stain – Process

The Results

One Step Trichrome - Gomori's The Result
One Step Trichrome - Gomori's The Result

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 minutes
Note: If heating Bouin’s in microwave, do not heat with slides in solution; heat Bouin’s, remove from MV, 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 min
5. Rinse in running water 5 min
6. One step blue/green solution 15 min
7. Straight to 1% glacial acetic acid 1 min
8. Rinse in running water
9. Dehydrate, clear and mount

Verhoeff's van Gieson Elastic Stain

Verhoeff's van Gieson 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

Verhoeff's van Gieson Elastic Stain – Process

The Results

Collagen – Red
Collagen – Red
Muscle – Yellow; Elastic Fibers – Black
Muscle – Yellow; Elastic Fibers – Black

The Protocol

Incubation times may vary per manufacturer guidelines.

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

 

Troubleshooting the Elastic Stain:

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 and 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.

Gomori's Reticulin Fiber

Reticulin fibers support the body and are common in liver, spleen and kidney. 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 vs. non-epithelial tumors.

Reticulin stains use silver and rely on the argyrophillic properties of the fibers. Argyrophillic cells can adsorb silver but cannot reduce it. Adsorb = surface based process whereas absorb = permeation through the tissue. Argyrophillic cells need a reducer/developer to convert the invisible silver salts to a visible metallic silver.

The Process

Gomori's Reticulin Fiber – The Process

The Results

Gomori's Reticulin Fiber – The Result

The Protocol

1. Bring slides to water
2. Oxidize in potassium permangenate 10 min
3. Rinse well in running water 3 min
4. Oxalic acid 1 min
5. Rinse well in running water 2 min
6. Sensitize in ferric ammonium sulfate 15 min
7. Rinse in distilled water 2 min
8. Impregnate in ammoniacal silver 2 min
9. Rinse in distilled water 1 min
10. Develop in 20% unbuffered formalin 1 min
11. Rinse well in running water 2 min
12. Tone in 0.1% gold chloride 3–5 min
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 min
17. Rinse briefly in water 15 sec
18. Dehydrate, clear and mount

 

Troubleshooting the Gomori’s reticulin:

Silver solution should be made fresh with distilled water and clean glassware (acid clean 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

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.

 

The Process

 

 

Movat Pentachrome – The Process

The Results

Movat Pentachrome – The Results

The Protocol

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

1. Bring slides to water
2. Alcian blue 20 min
3. Rinse well in running water 5 min
4. Alkaline alcohol 60 min
5. Rinse well in running water 10 min
6. Rinse in distilled water
7. Verhoeff’s elastic solution 12 to 15 min
8. Distilled water x 2
9. Ferric chloride 2 to 3 dips
10. Rinse in distilled water
11. Sodium thiosulfate 1 min
12. Rinse well in running water 5 min
13. Crocein scarlet – acid fuchsin 1:30 to 3 min
14. Destilled water x 2
15. .5% acetic acid water
16. 5% aqueous phosphotungstic acid x2 5 min each
17. .5% acetic acid water
18. Absolute alcohol x 3 Quick dips
19. Safran 15 min
20. Absolute alcohol x 2
21. Clear and mount

 

Troubleshooting the Movat Pentachrome

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 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.

Bibliography

Geoffrey Rolls. “101 Steps to Better Histology – A Practical Guide to Good Histology Practice”. Pathology Leaders/Articles

Shaikh, Dr. Imran, “Special Stains in Histopathology.”  Kem Hospital, 2012

Sheehan and Hrapchak, “Theory And Practice Of Histotechnology” Connective Tissue 2nd Edition 10: 180-200.

Culling, CFA, Allison R.T, Barr W.T., (1985) Cellular Pathology Technique, ed. 4. Butterworths, London, England.

Wikipedia – Ammoniacal Silver, Nov, 2013.

Russel, H.K.:  A Modification of the Movat Pentachrome Stain. Arch Pathol, 94: 187, 1972