International Journal of Orofacial Biology

: 2020  |  Volume : 4  |  Issue : 1  |  Page : 4--9

Connective tissue stains - A review

E Abigail Viola1, B Hindia2,  
1 Department of Oral Pathology and Microbiology, SRM Dental College, Chennai, Tamil Nadu, India
2 Private Practitioner, Meenakshi Ammal Dental College, Chennai, Tamil Nadu, India

Correspondence Address:
E Abigail Viola
Department of Oral Pathology and Microbiology, SRM Dental College, Chennai, Tamil Nadu


Over the years, a wide range of dyes has been used for histological staining methods. Most of these have been adapted from those used in the textile dyeing industry. The common histological stains appeared during the second half of the 19th century. Routinely, hematoxylin and eosin stain is widely used for histopathological examination of tissue sections. Although they have plethora of advantages, they lack to distinguish the various connective tissue components. This review aims to highlight the special stains used to describe the connective tissue elements which contribute in diagnosing the tumors of connective tissue origin.

How to cite this article:
Viola E A, Hindia B. Connective tissue stains - A review.Int J Orofac Biol 2020;4:4-9

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Viola E A, Hindia B. Connective tissue stains - A review. Int J Orofac Biol [serial online] 2020 [cited 2023 Jun 3 ];4:4-9
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Over the years, a wide range of dyes has been used for histological staining methods. Most of these have been adapted from those used in the textile dyeing industry. The dye with probably the greatest claim to antiquity is indigo. It is extracted from the Indigofera plant, and there is evidence of its use to dye cloth by the ancient Egyptians some 3000 years ago. Coincidentally, the Arabic term for indigo is Annil, from which the word Aniline derived. It is a substance extracted from indigo in the early 19th century. The aniline group of dyes had become the dominant force in the growth of the textile dyeing industry and histological stain technology. In the early 1860s, one-step staining had been used in which the excess stain was washed off in alcohol or water before the slide was mounted. In 1867, Schwartz introduced two-dye sequential staining interspersed with a simple washing stage. The technique was further refined when in 1869, Bottcher incorporated an alcohol differentiation step.[1]

The common histological stains appeared during the second half of the 19th century. These included fuchsin and carmine; methyl violet, alizarin still used for histochemical localization of calcium; methyl green; malachite green was until recently widely used to treat pond fish with parasitic diseases; indigo used as the basis for indigo techniques in the 1950s to localize histochemical activities of enzymes; and congo red still used to localize amyloid. Several of these dyes, such as rhodamine and malachite green, have been “rediscovered” and found new value in fluorescence microscopy. Orcein is still widely used for the localization of elastin. Weigert's elastin stain was introduced in 1898. Osmium tetroxide, which stains myelin, found a new lease of life as a contrast stain for transmission electron microscopy.[2] This review aims to highlight the different special stains used for the detection of various connective tissue components which are important as accurate diagnosis is the need of this era.

 Connective Tissue

Connective tissue is the most abundant tissue and can be found all over the body. It offers organs and other body structures a protective and supportive framework. Some cells make up connective tissue, which has a higher proportion of the extracellular matrix than other tissues.[3]

The three types of fibers present in the connective tissue matrix are reticular, collagenous, and elastic fibers.

Collagen protein is used to make reticular fibers, which are coated with glycoprotein. They form a delicate framework around nerve fibers, fat cells, lymph nodes, and smooth and skeletal muscle fibers, as well as a network around nerve fibers, fat cells, lymph nodes, and smooth and skeletal muscle fibersCollagenous fibers are made up of the protein collagen and they are the strongest of the three fiber types. Ligaments, tendons, cartilage, and bone all contain collagenous fibersElastic fibers which are made up of the protein elastin have the most flexibility of all the fiber types. Elastic fibers are found in the skin and the walls of blood vessels, and they allow the tissue to stretch.

 Reticular Fibers

The stains containing silver solutions are commonly used to demonstrate reticular fibers. These stains work by impregnating fibers with silver ions and then reducing those silver ions to their visible metallic form. Reticular fibers are argyrophilic in the manner that they can adsorb silver from solution but are unable to reduce it to visible metallic form without the aid of a reducing solution. There are many different silver stains that can be used to show reticular fibers such as Gomori, Snook, Gordon, and Sweets [Figure 1].{Figure 1}

The mechanism of action involves that a strong base (ammonium hydroxide) is added to an aqueous silver nitrate solution to form a silver diamine complex in an ammoniacal silver solution. Before the application of this complex, it is common practice to oxidize and sensitize the tissue. While the sensitizing agent (uranyl nitrate/dilute silver nitrate) initially binds to the tissue component of interest, oxidation (potassium permanganate/periodic acid) improves subsequent staining. The fibers are impregnated with silver ions from the ammoniacal silver solution, which replace the sensitizer in the metal–organic compound.[4] Further, the silver diamine complex is then reduced to a visible metallic form by the action of a reducing agent (formalin). Metallic silver is converted to metallic gold with the use of a toning reagent (gold chloride), which is more stable and provides better contrast and clarity. Excess gold chloride and unreduced silver are removed (sodium thiosulfate), and the tissue section is counterstained if desired. Counterstains such as nuclear fast red or light green are frequently used. The fibers are black and appear in a fine linear pattern.

 Gordon and Sweet's Staining Procedure

Deparaffinize sections with xylene then take through alcohols to waterOxidize in acidified potassium permanganate for 3 minRinse in distilled waterDecolorize with 2% oxalic acid for 1 minRinse in distilled waterMordant in 4% iron alum for 10 minRinse in distilled waterImpregnate in ammoniacal silver solution for 11 sRinse quickly in distilled waterImmediately reduce with 10% aqueous formalin for 2 minWash in running tap water for 2 minTone in 0.2% gold chloride for 2 minRinse in distilled waterFix with 2% aqueous sodium thiosulfate (hypo) for 2 minWash in water for 2 minCounterstain with neutral red for 2 minDehydrate, clear, and mount with DPX.

 Elastic Fibers

The Verhoeff-van Gieson stain, commonly used to demonstrate elastic fibers [Figure 2], is an example of a regressive staining method. The tissue section is initially overstained with a solution of hematoxylin-ferric chloride-iodine and then differentiated for optimal demonstration of elastic fibers.[5]{Figure 2}

Hematoxylin dye molecules are linked to the tissue components using ferric chloride and iodine as a mordant. They then convert hematoxylin to hematein by acting as oxidizers. The tissue-mordant-dye complex is then broken with a dilute solution of ferric chloride, allowing structures within the tissue to be distinguished. Elastic fibers retain dye molecules because they have a strong affinity for the iron-hematoxylin complex, whereas other structures dissociate dye molecules. The result is a stain reaction that is positive. Excess iodine and ferric chloride are removed from tissue sections using distilled water washes followed by sodium thiosulfate. As a counterstain, the van Gieson solution is used.[6]

The elastic fibers and nucleus appear as blue to black. Sometimes, the collagen fibers are also appreciated in red.

 Verhoeff-van Gieson Staining Procedure

Deparaffinize and hydrate slides to distilled waterStain in Verhoeff's solution for 1 h. Tissue should be completely blackRinse in tap water with 2–3 changesDifferentiate in 2% ferric chloride for 1–2 minStop differentiation with several changes of tap water and check microscopically for black elastic fiber staining and gray background. It is better to slightly under differentiate the tissue since the subsequent van Gieson's counterstain can extract the elastic stain somewhatWash slides in tap waterTreat with 5% sodium thiosulfate for 1 min. Discard solutionWash in running tap water for 5 minCounterstain in van Gieson's solution for 3–5 minDehydrate quickly through 95% alcohol and 2 changes of 100% alcoholClear in 2 changes of xylene for 3 min eachCoverslip with resinous mounting medium.


Muscle cells are characterized by the ability to contract, providing the movement necessary for locomotion of an organism, and movement of materials within an organism.

Three types of muscle tissue are identified based on their arrangement of actin and myosin fibers.[7]

Skeletal muscle cells are multinucleated and have striations, which are bands. Conventionally, these cells are arranged in bundles and attached to skeletal elements. They are responsible for locomotion and are controlled by motor neuronsWithin the interconnecting fibers of cardiac muscle cells, there are centrally located nuclei (intercalated discs). Only heart tissue contains cardiac muscle, which provides constant rhythmic contraction without the need for external stimulationSmooth muscle cells have a central nucleus and are spindle-shaped. Smooth muscle can be found in the skin as well as a variety of internal organs, such as the digestive and reproductive systems. They are in charge of involuntary movements such as peristalsis.

 Trichrome Stains

Masson's Trichrome Stain

With Masson's trichrome stain [Figure 3], Bouin's solution is used initially as a mordant to link the dye molecules to the tissue components of interest. Nuclei are stained with Weigert's hematoxylin, an iron hematoxylin, which is resistant to decolorization by subsequent acidic staining solutions. Application of Biebrich scarlet-acid-fuchsin stains all acidophilic tissue elements such as cytoplasm, muscle, and collagen. Subsequent treatment by phosphomolybdic/phosphotungstic acid serves as a decolorizer causing the Biebrich scarlet-acid-fuchsin to diffuse out of the collagen fibers while leaving the muscle cells red. Subsequent application of aniline blue will stain the collagen after which, 1% acetic acid is employed to properly differentiate the tissue section. Most tissue will contain an internal quality control, but the appendix, Fallopian tube, uterus, or small intestine may be used. By this staining, muscle appear as red, nucleus as black, and the surrounding collagen and other materials such as mucin appear as blue.[8]{Figure 3}

 Masson's Trichrome Staining Procedure

Deparaffinize and rehydrate through grades of alcoholWash in distilled waterFor formalin-fixed tissue, refix in Bouin's solution for 1 h at 56°C to improve staining quality, although this step is not absolutely necessaryRinse running tap water for 5–10 min to remove the yellow3. Stain in Weigert's iron hematoxylin working solution for 10 minRinse in running warm tap water for 10 minWash in distilled waterStain in Biebrich scarlet-acid-fuchsin solution for 10–15 min. Solution can be saved for future useWash in distilled waterDifferentiate in phosphomolybdic-phosphotungstic acid solution for 10–15 min or until the collagen is not redTransfer sections directly (without rinse) to aniline blue solution and stain for 5–10 min. Rinse briefly in distilled water and differentiate in 1% acetic acid solution for 2–5 minWash in distilled waterDehydrate very quickly through 95% ethyl alcohol, absolute ethyl alcohol (these steps will wipe off Biebrich scarlet-acid-fuchsin staining) and clear in xyleneMount with resinous mounting medium.

 Phosphotungstic Acid Hematoxylin

Although trichrome techniques will demonstrate muscle fibers, phosphotungstic acid hematoxylin (PTAH), or phosphotungstic acid-hematoxylin, is preferred for demonstrating cross-striations of skeletal muscle, which may be lost in certain muscle diseases [Figure 4].[9]{Figure 4}

Collagen and muscle fibers are demonstrated using a tungsten mordant provided by the phosphotungstic acid. This mordant binds hematein and stains selective tissue components blue, while the phosphotungstic acid is believed to stain other tissue components a red-brown. Tissue fixed in formalin is commonly postfixed in Zenker's solution, which is thought to intensify staining reactions. A section of skeletal muscle tissue can be used for quality control. Here, the muscle fibers appear in various shades of blue and collagen as red-brown.

 Phosphotungstic Acid Hematoxylin Staining Procedure

Deparaffinize sections if necessary and hydrate to distilled waterPour zinc chloride solution (10%) into plastic staining jar and set in 60°C. water bath for 10 min to equilibrate temperaturePlace slide in warmed zinc chloride solution (10%) and incubate for 20 min at 60°CDuring step 3, pour ferric ammonium sulfate aqueous solution into a second plastic staining jar and set in 60°C water bath for 10 min to equilibrate temperatureRinse slide in running tap water for 1 minRinse in distilled water for 1 minPlace the slide in warmed ferric ammonium sulfate aqueous solution and incubate for 5 min at 60°C. 8. During step 7, pour PTAH solution into a third plastic staining jar and set in 60°C water bath for 10 min to equilibrate temperatureRinse slide in running tap water for 2 minRinse in distilled water for 1 minPlace the slide in warmed phosphotungstic acid hematoxylin solution and incubate for 60 min at 60°C. 12. Differentiate section in 95% reagent alcohol. Check section using microscope for proper differentiationNote: graded alcohols will remove some stainDehydrate in 3 changes of absolute alcoholClear in 3 changes of fresh xylene or xylene substitute, and mount in DPX.


Oil-Red-O and Sudan Black B stain[10]

Oil-Red-O and Sudan Black B stains are used to demonstrate simple lipids [Figure 5] and [Figure 6]. Most stains demonstrate chemical interactions, but these “staining” techniques demonstrate physical processes.{Figure 5}{Figure 6}

(A) lipid solvent, such as propylene glycol, is used to dissolve the dye (Oil-Red-O or Sudan Black (B). other fat solvents, such as isopropanol, can be used, but they cause a loss of lipids. Dye molecules are more soluble in cellular lipid than in dye solvent and will migrate from the solvent into the tissue lipid, giving the tissue lipid a positive stain color.

After that, tissue sections are stained with a counterstain (hematoxylin/nuclear fast red). Fat-containing tissue serves as a source of quality control material. Fat appears red and nucleus appears blue in Oil-Red-O stain, whereas fat appears blue-black and nucleus appears red in Sudan Black B stain.

Oil-Red-O staining procedure

Mount cryostat-cut sections of clean slides and fix in 10% buffered neutral formalin for 15 min. Formalin-fixed frozen sections may be floated in water and attached to albuminized slides. Allow to thoroughly dry (1to 2 h) to assure paper adherence. Fixation for touch preparations and material from cytology is not necessaryRinse briefly with distilled waterA few dips in 60% isopropyl alcoholOil-Red-O working solution for 20 minA few dips in 60% isopropyl alcoholRinse with four changes of distilled waterCounterstain with hematoxylin stain for 1 minRinse in three changes of distilled waterBlue hematoxylin in 0.3% sodium borate for 15 sRinse with four changes of distilled waterMount with DPX.

 Sudan Black B Staining Procedure

Deparaffinize and hydrate to 70% alcohol. When staining frozen sections, begin at step 3Stand slides on end and allow to dryPlace in Sudan Black B solution for 1 to 3 h. Check after 1 h but first dip in 70% isopropyl alcohol. When staining frozen sections, stain for 20 min at room temperatureRinse thoroughly in two changes of 70% isopropyl alcoholWash with six changes of distilled waterNuclear fast red solution for 3 minRinse in two changes of distilled waterMount with DPX.


Connective tissue stains have been used extensively for the diagnosis of tumors of varying origins. Understanding these staining techniques not only aids us in performing our staining procedures effectively but also can facilitate the innovation of new methods.

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Conflicts of interest

There are no conflicts of interest.


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