ホーム • Knowledge Pathway • チュートリアル • Special Stains – Which One, How and Why? Part II: Connective Tissue Special Stains – Which One, How and Why? Part II: Connective Tissue Carolyn Doan HT (ASCP) Definition of "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. Image 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 Image The Results Image 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 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. 10-15 mins 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 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% Glacial Acetic Acid (GLAA). 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 Image The Results Image Image 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 solution; heat Bouin's, remove from microwave, place slides in Coplin jar, seal with lid and incubate. 10-15 mins 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 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 Image The Results Image Colágeno: rojo Image Músculo: amarillo; Fibras elásticas: negro 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 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. 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. Image 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 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. The Process Image The Results Image 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 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: Image 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 Image The Results Image The Protocol Incubation times and reagent sequence may vary per manufacturer, but results are similar. 1. Bring slides to water 2. Alcian blue 20 mins 3. Rinse well in running water 5 mins 4. Alkaline alcohol 60 mins 5. Rinse well in running water 10 mins 6. Rinse in distilled water 7. Verhoeff's elastic solution 12-15 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 mins 13. Croceine scarlet – acid fuchsin 90 sec - 3 min 14. Distilled water x 2 15. .5% acetic acid water 16. 5% aqueous phosphotungstic acid x 2 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 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. About the presenter Carolyn Doan , HT (ASCP) Carolyn Doan received her ASCP registration in 1969 after completing classes at Georgia State University and St Joseph school of Histotechnology in Atlanta, Georgia. For 40 years she shared her passion for histology in research (at Yerkes Primate Research Center), in the clinical world (managing the Pathology Department at Florida Hospital in Orlando and serving on the board of the Florida State Licensure Task Force and as president of the Florida State Society for Histotechnology), and in industry (as a Sales executive, Marketing Manager and North American staining sales specialist). Since her retirement in 2013, she founded Creative Histology Consulting. References Bibliography Rolls, Geoffrey. “101 Steps to Better Histology – A Practical Guide to Good Histology Practice”. Knowledge Pathway/Training Resources. Shaikh, Imran. “Special Stains in Histopathology” Kem Hospital, 2012. Sheehan, D. “Theory and Practice of Histotechnology.” Connective Tissue. 2nd edition, 10: 180-200. Culling, C., et.al. Cellular Pathology Technique, edition 4. Butterworths, London, England 1985. Wikipedia – Ammoniacal Silver, Nov 2013. Russel, H.K. A Modification of the Movat Pentachrome Stain. Arch Pathol, 94: 187, 1972. Leica Biosystems Knowledge Pathway content is subject to the Leica Biosystems website terms of use, available at: Legal Notice. The content, including webinars, training presentations and related materials is intended to provide general information regarding particular subjects of interest to health care professionals and is not intended to be, and should not be construed as, medical, regulatory or legal advice. The views and opinions expressed in any third-party content reflect the personal views and opinions of the speaker(s)/author(s) and do not necessarily represent or reflect the views or opinions of Leica Biosystems, its employees or agents. 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