On the mechanism of Verhoeff's elastica stain: A convenient stain for myelin sheaths

Summary Verhoeff (1908) recommended an iron-hematein formula containing Lugol's solution for demonstration of elastic tissue; sections are differentiated until desired staining patterns are obtained. Verhoeff's stain colored a variety of tissue structures and showed higher substantivity for myelin sheaths than for elastin. Addition of HCL or omission of Lugol's solution decreased or abolished coloration of pseudo-elastica and thus enhanced selectivity for elastin. Substitution of Fe⁺⁺ for Fe⁺⁺⁺ abolished dye binding by elastin.A review of chemical data indicated interaction of components of Lugol's solution with the dye. Hematein and Fe⁺⁺⁺ form a variety of cationic, anionic and non-ionic chelates; the ratio of these compounds changes with time. Dye binding apparently occurs mainly via van der Waals forces and hydrogen bonds.Verhoeff's elastica stain is definitely not specific for elastin and is inferior to orcein and resorcin-fuchsin because of the required differentiation with its inherent bias to produce patterns which conform to expectations. However, Verhoeff's elastica stain is far superior to other metal-hematein technics for myelin sheaths. The combined Verhoeff-picro-Sirius Red F3BA stain can be performed in 30 min and does not require differentiation. It is therefore suggested to reclassify Verhoeff's elastica stain as a method for myelin sheaths.     

A combination Verhoeff's elastic and Masson's trichrome stain for routine histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeff's technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

Improved iron-hematoxylin stain for elastic fibers

A modification of Verhoeff's elastic tissue stain with connective tissue counterstaining is described. The modified procedure requires no differentiation of the elastic fibers, thus eliminating the problem of over- or understaining of elastic fibers. The procedure is easy to perform and yields consistently good results with sharply defined elastic fibers that are easily distinguishable from other connective tissue elements. It is recommended for routine use, particularly when photomicrography is desired.     

Modified elastic tissue-Masson trichrome stain

A combined elastic tissue-Masson technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeff's iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Simultaneous demonstration of connective tissue elastica and fibrin by a combined Verhoeff's elastic-Martius-scarlet-blue trichrome stain

A method for the routine combined demonstration of elastica, connective tissue in general, and fibrin is described. Elastica, stained blue-black by Verhoeff's iron hematoxylin, is contrasted with muscle and collagen, stained red and blue or green respectively, by a modification of the Martius-scarlet-blue (MSB) trichrome for fibrin of Lendrum et al. The MSB technique selectively stains fresh, mature and old fibrin orange-yellow, red and blue respectively; the Masson trichrome does not distinguish between erythrocytes and fibrin. Nuclei are stained at the same time as the elastica. The technique takes approximately one and a half hours and is ideal for the study of connective tissue and vascular pathology, especially the necrotizing vasculitides.     

Palladium chloride as a stain for elastin at the ultrastructural level.

Palladium chloride in aqueous solution stains elastic fibers in thin sections of Epon-embedded tissues. When palladium chloride is used with a lead citrate counterstain, high contrast sections with gray to black elastic fibers are obtained. The stain was tested on newborn and adult mammalian tissues and on adult tissues from lower animals. Sections were mounted on stainless steel grids, stained with 1% palladium chloride solution for 5 to 15 min, rinsed thoroughly, and counterstained with lead citrate for 7 min. Palladium chloride staining solution is stable for several months at room temperature and if the stain is filtered immediately before use, contamination of sections is not a problem. Chemical studies indicate that palladium binds directly to purified bovine ligamentum nuchae elastin and that this binding is not affected by glutaraldehyde fixation or by sodium borohydride reduction of elastin. Osmium post-fixation of glutaraldehyde-fixed elastin did significantly lower the amount of palladium bound. Palladium was shown to be chemically bound to sites on the elastin and not weakly associated. The nature of these sites is discussed.     

Iron gallein elastic method---a substitute for Verhoeff's elastic tissue stain.

A method for staining elastic fibers in formalin fixed, paraffin embedded sections is described. After deparaffinizing and dehydration, sections are stained for 30 minutes in a solution prepared by mixing equal parts of 1% gallein dissolved in ethylene glycol and absolute alcohol (1:4), and 1.16% aqueous ferric chloride in 1% hydrochloric acid. The sections are washed in water and then differentiated in 2% ferric chloride for 2 minutes. After washing in water, the sections are counterstained with a variant of Van Gieson's picric acid-acid fuchsin for 1 minute. The results are similar to Verhoeff's elastic stain with elastic fibers staining black. An advantage to this staining procedure is that visually controlled differentiation is not necessary.     

Orcein, collastin and pseudo-elastica: a re-investigation of Unna's concepts.

Orcein has been recommended for identification of elastin. Since other traditional elastica stains proved to be unspecific, it was deemed of interest to determine the selectivity of orcein and to review pertinent literature. Orcein was employed as a textile dye in ancient Egypt and was used for dyeing of wool and silk until the early 20th century. It was introduced into histological technic in 1878 as a stain for cytoplasm. Unna recommended it for demonstration of elastic tissue in 1890 and retracted claims for its specifity in 1894 because orcein colored also certain collagen fibers. Unna suggested the term collastin for collagen fibers which share the affinity of elastin for acid orcein. Other orcein solutions were used as selective stains for collagen. In histochemical studies, the staining properties of resorcin-fuchsin and orcein were very similar; elastin and various collagen fibers were strongly colored. Unna's collastin is apparently identical with the pseudo-elastica described in sections stained with resorcin-fuchsin. Both dyes react with meshworks of fine fibers, embryonic, experimentally or pathologically altered collagens. It is suggested to use the term collastin, instead of pseudo-elastica, for collagenous fibers which bind the traditional elastica stains.     

Orcein,collastin and pseudo-elastica: a re-investigation of Unna's concepts

Summary Orcein has been recommended for identification of elastin. Since other traditional elastica stains proved to be unspecific, it was deemed of interest to determine the selectivity of orcein and to review pertinent literature.Orcein was employed as a textile dye in ancient Egypt and was used for dyeing of wool and silk until the early 20th century. It was introduced into histological technic in 1878 as a stain for cytoplasm. Unna recommended it for demonstration of elastic tissue in 1890 and retracted claims for its specifity in 1894 because orcein colored also certain collagen fibers. Unna suggested the term collastin for collagen fibers which share the affinity of elastin for acid orcein. Other orcein solutions were used as selective stains for collagen.In histochemical studies, the staining properties of resorcin-fuchsin and orcein were very similar; elastin and various collagen fibers were strongly colored. Unna's collastin is apparently identical with the pseudo-elastica described in sections stained with resorcin-fuchsin. Both dyes react with meshworks of fine fibers, embryonic, experimentally or pathologically altered collagens. It is suggested to use the term collastin, instead of pseudo-elastica, for collagenous fibers which bind the traditional elastica stains.     

Notes on Technic: Mahon's Myelin Stain for Celloidin-Embedded Nervous Tissue

Two methods commonly used to stain myelin sheaths are Kluver and Barrera's luxol fast blue (Kluver and Barrera 1953) and Weil's iron hematoxylin (Weil 1928). Both require differentiation of the stain; in addition, the Kluver-Barrera method specifies 16-24 hour staining. A third method for the selective staining of myelinated axons is that of Mahon (1938), which was introduced for use with paraffin-embedded autopsy tissue. The procedure possesses two distinct advantages since it requires: (1) no differentiation of the stain and (2) only 1 hour staining. Loyez's (1910) myelin stain for celloidin embedded tissue is similar to Mahon's but calls for long staining followed by differentiation. This report describes the application of Mahon's method to celloidin-embedded experimental tissue and emphasizes its utility for staining tissues to be used for reconstructing microelectrode penetrations (fig. 1) and for demonstrating the effect of experimental lesions (fig. 2).     

Identification of a uterine elastase in the pregnant rat uterus

During development of the pregnant rat uterus there is a several fold increase in elastin content. Using Verhoeff's elastic fiber stain, we have shown that a significant proportion of these elastin fibers are in the extracellular matrix of the myometrium. They do not appear as an organized structure but rather in a variety of partially extended, random configurations. An elastase was identified in both the pregnant and the postpartum uterus. Partial characterization of the enzyme indicated that it is a serine protease with a molecular weight around 24,500 and a pH optimum of 8.5. In addition to the enzyme, relatively high levels on an elastase inhibitor were found in the uterine extracts. The inhibitor did not inhibit trypsin, indicating that it was not alpha-1-antiprotease. The data suggest that the elastase and inhibitor are uterine tissue derived and perhaps important in the normal remodeling process of uterine connective tissue.     

Demonstration of Elastic Tissue and Iron or Elastic Tissue and Calcium Simultaneously

For the concomitant demonstration of iron and elastic tissue Perls' test solution was used, followed by Verhoeff's stain or Gomori's aldehyde fuchsin. When Perls' and Verhoeff's stain were used in sequence, the iron deposits were greenish blue and the elastic lamellae were black. When Perls' test solution was combined with aldehyde fuchsin the iron deposits were blue and elastic tissue purple. Calcium salts and elastic tissue were demonstrated concomitantly by using von Kossa's method followed by Gomori's aldehyde fuchsin. With such combined staining, the calcium salts appeared brownish black and elastic tissue purple. With these procedures, it was possible to see the exact relationship of calcium and iron deposits to the elastic tissue.     

A combined elastic, fibrin and collagen stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified Verhoeff's elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by lissamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsin, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

Ethidium bromide: a nucleic acid stain for tissue section

The phenanthridinium dye, ethidium bromide (EB), selectively intercalates into double-stranded regions of nucleic acids with a large and specific increase in fluorescence. When used for the staining of fixed tissue sections, the dye stains cellular nuclei with excellent resolution of microscopic detail. In some fixed tissues, particularly pancreatic acini, cytoplasm stains intensely and this staining can be abolished by digestion with trypsin and ribonuclease. The orange fluorescence of EB can be easily distinguished from the green fluorescence of fluorescein and EB is thus an excellent counterstain for immunofluorescence. Ethidium bromide is a useful and practical stain for the fluorescence microscopy of tissue sections and, in combination with enzymatic digestion of RNA, provides a simple way to differentially localize DNA and RNA.     

Bismuth Hematoxylin Stain for Arginine Residues

Bismuth ions complex with hematoxylin oxidized by sodium iodate to form a dark blue dye that stains structures with high arginine content. In citrate buffer at pH 5.2, staining is confined to cell nuclei and myelin sheaths. Extraction of nucleic acids has little effect on the stain. Blockade of the guanidino groups of arginine completely abolishes staining.     

Application of current chemical concepts to metal-hematein and -brazilein stains

Current chemical concepts were applied to Weigert's, M. Heidenhain's and Verhoeff's iron hemateins, Mayer's acid hemalum stain and the corresponding brazilein compounds. Fe bonds tightly to oxygen in preference to nitrogen and is unlikely to react with lysyl and arginyl groups of proteins. Binding of unoxidized hematoxylin by various substrates has long been known to professional dyers and was ascribed to hydrogen bonding. Chemical data on the uptake of phenols support this theory. Molecular models indicate a nonplanar configuration of hematoxylin and brazilin. The traditional quinonoid formula of hematein and brazilein was revised. During chelate formation each of the two oxy- groups of the dye shares an electron pair with the metal and contributes a negative charge to the chelate. Consequently, the blue or black 2:1 (dye:metal) complexes are anionic. Olation of such chelates affects the staining properties of iron hematein solutions. The color changes upon oxidation of hematoxylin, reaction of hematein with metals, and during exposure of chelates to acids can be explained by molecular orbital theory. Without differentiation or acid in dye chelate solutions, staining patterns are a function of the metal. Reactions of acidified solutions are determined by the affinities of the dye ligands. Brazilein is much more acid-sensitive than hematein. This difference can be ascribed to the lack of a second free phenolic -OH group in brazilein, i.e. one hydrogen bond is insufficient to anchor the dye to tissues. Since hematein and brazilein are identical in all other respects, their differences in affinity cannot be explained by van der Waals, electrostatic, hydrophobic or other forces.     

Quantitative histochemistry of myelin using Luxol Fast Blue MBS

Synopsis The amount of Luxol Fast Blue MBS in the band of Genarri was measured with two types of scanning microdensitometer and the optical density determined. The amount of stain measured was proportional to the section thickness employed, thus demonstrating that the dye has stoichiometric properties in tissue sections. Blocks of tissue treated with phospholipid solvents showed an increased uptake of stain, suggesting that phospholipids are not a primary substrate for the dye in myelin staining.The dye may, therefore, be used to quantify myelin in tissue sections.     

Suggested Counterstains for Davenport Reduced Silver Preparations of Peripheral Nerves

—Peripheral nerves which have been fixed in a mixture of formaldehyde and acetic acid and stained according to the method of Davenport can be successfully counterstained for demonstration of myelin sheaths and stroma. After mounted sections have been silvered, reduced and toned, the coating of nitrocellulose is removed by passing thru two changes of acetone. Following brief washes in 100,95,85 and 75% alcohols they are stained in an acidified aqueous solution of azo carmine for 30 to 60 minutes. Excess azo carmine is extracted with anilin alcohol followed by acetic alcohol after which the sections are mordanted for 15 to 60 minutes in a 5% aqueous solution of phosphotungstic acid. Without washing they are transferred to a stain mixture of either anilin blue and orange G (acidified) or light green and orange G (acidified) where they remain from 1 to 5 hours. After destaining in 95% alcohol and dehydration in absolute alcohol the sections are mounted in dammar. Result: axons stain black; sheath and fibroblast nuclei, red; myelin sheaths, orange; and connective tissue, blue or green. When the counterstains are applied to ganglia, cytological details of individual cells are demonstrated.     

Simultaneous Demonstration of Connective Tissue elastica and Fibrin by a Combined Verhoeff's Elastic-Martius-Scarlet-Blue Trichrome Stain

A method for the routine combined demonstration of elastica, connective tissue in general, and fibrin is described. Elastica, stained blue-black by Verhoeff's iron hematoxylin, is contrasted with muscle and collagen, stained red and blue or green respectively, by a modification of the Martius-scarlet-blue (MSB) trichrome for fibrin of Lendrum et al. The MSB technique selectively stains fresh, mature and old fibrin orange-yellow, red and blue respectively; the Masson trichrome does not distinguish between erythrocytes and fibrin. Nuclei are stained at the same time as the elastica. The technique takes approximately one and a half hours and is ideal for the study of connective tissue and vascular pathology, especially the necrotizing vasculitides.     

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.