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.     

An Orcein-Oil Red O Stain for Concomitant Demonstration of Elastic Tissue and Lipid

Orcein, 0.5% in 50% isopropanol, 0.5-1 hr, followed by saturated oil red O in isopropanol diluted 3:2 with distilled water, 10-15 min, was used to demonstrate lipids and elastic tissue simultaneously in 10 μ frozen sections of formalin-fixed aortas of the wild African buffalo, showing atherosclerotic lesions. A comparison was made with the oil red O-aldehyde fuchsin (AF) method of Kwaan and Hopkins (Stain Techn., 39: 123-5, 1964) and the resorcin fuchsin (RF)-oil red O method of Lillie (Histopathologic Technic and Practical Histochemistry, McGraw-Hill, 1954), but both gave marked background staining by AF or RF that obscured the smaller deposits of lipid. Sudan IV could be substituted for oil red but did not demonstrate many of the finest deposits of lipids. Sudan black, in combination with orcein, AF or RF, was very satisfactory for demonstrating lipids but obscured many elastic fibres. Sudan dyes I, II, III, brown, blue, and green, with orcein, AF or RF, showed less contrast between lipids and elastic tissue or failed to stain the lipids adequately.     

Elastic Tissue Staining

It has been found that the addition of dextrin to samples of crystal violet and basic fuchsin employed in the prepararation of the elastic tissue stain after the technic of Weigert makes more sure a satisfactory final product. A modification of the original Weigert technic employing crystal violet or a mixture of crystal violet and basic fuchsin is offered as providing a better color contrast both visually as well as photographically. Crystal violet alone affords a bright greenish-yellow elastin while the addition of basic fuchsin results in a darker stain shading into dark blue as the proportion of basic fuchsin is increased.     

Modified Elastic Tissue-Masson Trichrome Stain

A combined elastic tissue-Massou 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 Verhoeffs 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.     

Structure and Development of the Notochord ‘Elastica Externa’ and Nearby Components of the Elastic Fibre System of Agnathans

Between 15 days and 3 months in age, the ‘elastica externa’ of the notochord sheath of larval lampreys develops from patches of moderately dense and amorphous material into a thick, continuous and electron-dense layer. In both lampreys and hagfish, this layer stains strongly with Verhoeff's elastic stain and aldehyde fuchsin and is penetrated by collagen fibrils on both its outer and inner boundaries. Peroxidase labelling using an antibody raised against human elastin specifically labels both the notochord ‘elastica externa’ and the elastic fibre system of lampreys. The diameters of the microfibrils (10–13 nm) of the oxytalan, elaunin and elastic fibres of lampreys and hagfish are the same as those of higher vertebrates. The connective tissue immediately dorsal and ventral to the notochord of lampreys contains mainly oxytalan fibres in very young ammocoetes, a combination of oxytalan, elaunin and elastic fibres in older ammocoetes, and predominantly elastic fibres in adult lampreys. While the region of the endomeninx at the base of the spinal cord contains almost exclusively oxytalan fibres in young ammocoetes, it also possesses numerous elastic fibres in adult lampreys. These findings indicate that, as in higher vertebrates, the elastic fibres of lampreys develop from oxytalan fibres via elaunin fibres.     

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Klig-man's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.     

A Combination Verhoeffs 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 Verhoeffs 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.     

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 am counterstained with a variant of Van Girson'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.     

Staining properties of aldehyde fuchsin analogs.

This investigation was designed to clarify the role of the aldehyde component of aldehyde fuchsin in its staining reactions. Several aldehyde fuchsin analogs were prepared by using different aldehydes. The staining quality of these analogs and pararosaniline-HCl was compared with that of aldehyde fuchsin prepared with paraldehyde in the usual way. The major findings of this investigation include: 1) Aldehyde fuchsin staining of nonoxidized pancreatic B cells requires a stain prepared with either paraldehyde or acetaldehyde. 2) An aldehyde moiety is required for aldehyde fuchsin staining of strong tissue anions. 3) Staining of elastic tissue with aldehyde fuchsin analogs resembles staining of strong tissue anions more than staining of nonoxidized pancreatic B cells. Possible reaction mechanisms of aldehyde fuchsin with tissue substrates are discussed.     

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.     

Iron and Aluminum Lakes of Gallo Blue E As Nuclear and Metachromatic Mucin Stains

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This adoring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast ells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent wed and the amount of Al²⁺ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

Iron and aluminum lakes of Gallo blue E as nuclear and metachromatic mucin stains.

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This coloring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast cells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent used and the amount of Al3+ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

A Selective Stain for Elastic Tissue (Orcinol-New Fuchsin)

A selective stain for elastic tissue (designated orcinol-new fuchsin) is described. Two grams of new fuchsin (C.I. No. 678) and 4 gm of orcinol (highest purity) are added to 200 ml of distilled water and the solution boiled for 5 min. Then 25 ml ferric chloride solution (U.S.P. IX) are added and the solution is boiled 5 min longer. The precipitate is collected and dissolved in 100 ml 95% ethanol. This is the staining solution. Sections are deparaffinized and brought to absolute ethanol, stained for 15 min at 37 °C with orcinol-new fuchsin, differentiated for 15 min in 70% ethanol, dehydrated, cleared and covered as usual.     

Acid Orcein-Iron and Acid Orcein-Copper Stains for Elastin

Paraffin sections of formol-fixed tissues stained 4-18 hr in 70% alcohol containing 1% orcein and 1% of concentrated (12 N) HCl by volume yield the familiar purple brown elastin and red nuclei on a pink background. When sections so stained are transferred directly from the stain to 70% alcohol containing 0.02% ferric chloride (FeCl₃·6 H₂O) or 0.02% copper sulfate (CuSO₄·5 H₂O) for a 15 sec to 3 min period, elastin coloration is changed to black or reddish black and chromatin staining to reddish black. The procedure can be counterstained with picro-methyl blue to yield blue collagen and reticulum or with our flavianic acid, ferric chloride, acid fuchsin mixture to give deep yellow background and deep red collagen.     

The Application of Miller's Elastic Stain to Glycol Methacrylate Tissue Sections

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.     

Staining Myelin, Elastic Fibers and Nuclei with Iron Hematoxylin

Two iron hematoxylin staining procedures were developed. Both use stable stock solutions and can be prepared volumetrically. The nuclear stain is progressive but differentiation is required for myelin sheath and elastic tissue staining. Histochemical procedures demonstrated that acid, hydroxyl, and aldehyde groups play no role in the staining but amine groups are essential. With both types of stains neither electrostatic bonding nor hydrogen bonding is essential but the nature of the union between tissue and the iron hematoxylin complex was not determined.     

Advantages in the Use of Aldehyde Fuchsin with Van Gieson's Picro-Fuchsin Counterstaining for Differentiating Bone and Cartilage

Specimens of bone were fixed in 10% neutral phosphate-buffered formalin or in Bouin's fluid and decalcified in 10% formic acid buffered with 10% sodium citrate. Materials were embedded in paraffin and 4-5 μ sections attached to slides were oxidized with 0.5% KMnO₄, decolorized in 1% oxalic acid, stained with aldehyde fuchsin, and counter-stained with Van Gieson's picro-fuchsin. Sections were dehydrated, cleared and mounted in a synthetic resin. Microscopically, the differentiation between bone and cartilage was seen as red and purple respectively, with connective tissue red; muscle and erythrocytes, yellow; and elastic fibres purple. The areas occupied by bone, cartilage and erythrocytes could be compared, and also the depth to which cartilage extended into the ossified sites. The advantages of this staining combination are: good contrasts in colour, ease of applying the stain, and virtual self-differentiation of the staining solutions.     

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.     

Metallic Lakes of the oxazines (Gallamin Blue,Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2–3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3–5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

The oxidative generation of sulfonic acid groups in neuromelanin and lipofuscin in the human brain

Sulfonic acid groups were oxidatively generated in the soluble lipid-free lipofuscin component of neuromelanin of human substantia nigra and in lipofuscin of human inferior olive. Exposure of these oxidized, intraneuronal pigments to low pH Alcian blue or aldehyde fuchsin demonstrated an intensity of staining that related to the type of oxidant and the conditions of its use. Utilization of the following oxidants generated increasingly strong staining reactions as signified by the following sequence; periodic acid under mild conditions, bromine in carbon tetrachloride, hydrogen peroxide, periodic acid under drastic conditions, potassium permanganate followed by oxalic acid, hydrogen peroxide followed by bromine in carbon tetrachloride, potassium permanganate followed by metabisulfite or bisulfite, and performic acid. Neither Alcian blue nor aldehyde fuchsin revealed oxidatively generated aldehyde as judged by 1) their failure or near failure to stain inferior olive lipofuscin following mildly applied periodic acid, and 2) the increase in staining intensity, from moderate to strong, displayed by the soluble lipid-free lipofuscin component of neuromelanin and by inferior olive lipofuscin when potassium permanganate was followed by a rinse with metabisulfite or bisulfite in place of one with oxalic acid.