A comparative study of PAS-phosphotungstic acid-Diamine Supra Blue FGL and immunological reactions for type I collagen.

Immunohistochemical techniques proved valuable in histological studies of various types of collagens. However drawbacks include non-specific reactions of antibodies, masking of antigens, and the high cost of antibodies. This study was undertaken to ascertain the specificity of the PAS-phosphotungstic acid-Diamine Supra Blue FGL (PAS-PTA-DSB-FGL) reaction for type I collagen, differentiating it from other collagens. Duplicate series of methacarn-fixed sections of various tissues were treated with the PAS-PTA-DSB FGL reaction and the peroxidase-antiperoxidase (PAP) technique for type I collagen and the staining patterns were compared. Fibers binding the blue dye were found only at sites reacting with antibodies against type I collagen. These observations indicate that the PAS-PTA-DSB FGL procedure is suitable for visualization of type I collagen, e.g. in screening of large series of sections and in the practice of surgical and autopsy pathology.

Are picro-dye reactions for collagens quantitative? Chemical and histochemical considerations.

Previous studies of picro-dye reactions demonstrated wide variations in the binding of different dyes. Picro-Sirius Red F3BA was recommended because it colors all collagens intensely and is suitable for polarization microscopy. Recent publications on quantitative uses of this stain were surprising. To obtain further information on the chemical mechanisms of dye binding by proteins, 94 sulfonated azo dyes were tested under the conditions of the picro-Sirius Red F3BA reaction. Reaction patterns varied widely, from failure to compete successfully with picrate ions for binding sites to strong coloration of all tissue structures. Only a few dyes stained collagen, reticulum fibers and basement membranes intensely and selectively. The reactivity of dyes was determined by their molecular configuration and the nature and position of substituents. Correlation with physico-chemical data showed that dye binding is due to non-ionic interactions, i.e. van der Waals and dispersion forces and hydrophobic bonding. Coulomb forces do not impart affinity - increasing sulfonation actually decreases dye uptake - but draw dyes within reach of non-ionic sites. Bound dyes form aggregates with additional dye ions; the aggregation number can range from 2 to many powers of 10. Clearly, dye binding by proteins is not stoichiometric.

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.

A comparative study of myosins and prekeratin in epithelial cells of methacarn-fixed tissues.

Around the turn of the century, tonofibrils and contractile myofibrils were observed within the same cells. These findings have been largely forgotten. To clarify the topical relations of these proteins in epithelial cells, duplicate sections of methacarn-fixed human and canine tissues were treated with the tannic acid-phosphomolybdic acid (TP)-Levanol Fast Cyanine 5RN reaction for myosins and the PAP technic for prekeratin, respectively. In bronchi, lingual and sweat glands, liver and pancreas, myosin was confined to the terminal bar-terminal web system, including pericanalicular layers. Prekeratin occurred throughout the epithelium of bronchi and ducts; secretory cells showed little or no reaction. Observations on myosin in kidney confirmed data by Harper et al. (1970). The PAP technic colored transitional epithelium and collecting tubules intensely; convoluted tubules did not react. Staining of segments of Henle's loops varied from case to case. Both reactions colored thymic epithelial cells. In myoid cells of Hassall's corpuscles myosin was gradually replaced by prekeratin and keratin. Basal cells of epididymis reacted strongly with the PAP technic, but did not contain myosin. Prekeratin is apparently identical with epidermin, whose composition and structure were well known in the 1950's. Epidermin undergoes chemical changes as cells move from the stratum basale to the stratum corneum. According to DAKO, the antibodies used in this study were prepared with prekeratin extracted from stratum corneum. Data in the literature and observations in this investigation indicate that some samples of antibodies do not react with all tonofilaments.(ABSTRACT TRUNCATED AT 250 WORDS)

A review of light, polarization and fluorescence microscopic methods for amyloid.

Traditional technics for amyloid are not always dependable. Therefore, several reactions, which do not require differentiation, were developed in this laboratory. This review describes technics that proved suitable for diagnostic pathology, including polarization and fluorescence microscopy. Effects of fixation on the reactivity of amyloid are also considered. The chemical mechanism of the alkaline Congo red, Mesitol WLS-Congo red and Phorwhite BBU reaction, and of a modified thioflavine T stain are reviewed briefly. Problems encountered with other methods, which cannot be recommended for diagnostic pathology, are outlined.

On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions.

Formalin has been recommended as an innocuous fixative for immunohistochemistry. However, several studies demonstrated impairment or blocking of antigenic activity of certain proteins. Formalin fixation was discovered accidentally by F. Blum in 1893 and its deleterious effects on various tissue structures were discussed extensively during the following decades. More recently, some authors assumed that formaldehyde bound to tissues can be largely or completely removed by washing and dehydration. According to chemical data, formaldehyde forms highly reactive methylols with uncharged amino groups. Such methylol groups yield methylene bridges with suitably spaced amides, arginine and aromatic amino acid sidechains. Only loosely bound formaldehyde is removed by washing for several hours. Residual bound formaldehyde cannot be dislodged by washing for weeks, but some formaldehyde is gradually removed when tissues are stored in water for an extended number of years. Methylene crosslinks resist treatment with high concentrations of urea, and can be broken only by drastic hydrolysis. It appears unlikely that such firmly bound formaldehyde is removed by conventional washing and dehydration procedures used in histochemistry. The superiority of methacarn, alcohol or acetone over formaldehyde fixation for immunohistochemical demonstration of prekeratin, myosin, type I and type IV collagen, laminin and fibronectin can be ascribed to the irreversible alterations of tissue proteins by formaldehyde.

Current chemical concepts of acids and bases and their application to anionic ("acid") and cationic ("basic") dyes.

In biomedical studies, dyes are divided into "acid" and "basic" dyes. This classification cannot be reconciled with current chemical definitions of acids and bases. Brönsted-Lowry acids are compounds that can donate protons; bases are proton acceptors. The definition of acids and bases is independent of the electric charge, i.e. acids and bases can be neutral, anionic or cationic. Reactions between acids and bases result in formation of new acid-base pairs. Lewis acids and bases do not depend on a particular element, but are characterized by their electronic configurations. Lewis bases are electron donors; Lewis acids are electron acceptors. This classification is also unrelated to the electric charge. Lewis acids and bases interact by formation of coordinate covalent bonds. In histochemistry and histology, dyes containing -SO3-, -COO- and/or -O- groups are classified as "acid" dyes. However, such compounds are electron pair donors and hence Brönsted-Lowry and Lewis anionic bases. Dyes carrying a positive charge are termed "basic" dyes. Chemically, many cationic dyes are Lewis acids because they can add a base, e.g. OH-, acetate, halides. The hypothesis that transformation of -NH2 into ammonium groups imparts "basic" properties to dyes is untenable; ammonium groups are proton donors and hence acids. Furthermore, conversion of an amino into an ammonium group blocks a lone electron pair and the color of the dye changes drastically, e.g. from violet to green and yellow. It appears therefore highly unlikely that ammonium groups are responsible for binding of cationic ("basic") dyes. In histochemistry, it is usually not of critical importance whether anionic or cationic dyes are chemically acids or bases.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of early lesions of muscle by infrared fluorescence microscopy.

Previous investigations demonstrated decrease or loss of IR fluorescence in certain lesions of smooth and striated muscle. These investigations were extended to diseases of skeletal muscle. Sections were stained and photographed as described (Puchtler et al. 1980). Various diseases caused significant changes of IR fluorescence. Often, IR fluorescence demonstrated lesions in muscle fibers which appeared unremarkable in direct light and/or conventional darkfield microscopy. The patterns of IR fluorescence varied widely, even within groups of diseases, e.g. muscular dystrophies. However, owing to the heterogeneity of the available material, it is not yet possible to draw conclusions concerning the diagnostic significance of different IR fluorescence patterns. Hence this report should be regarded only as a pilot study.

Application of thiazole dyes to amyloid under conditions of direct cotton dyeing: correlation of histochemical and chemical data.

The fluorescent brightening agent Phorwhite (Blankophor) BBU imparts intense selective fluorescence to amyloid, but this modern reagent is no longer readily available on the biological dye market. Conventional Thioflavine S and T stains require differentiation and are not specific. To improve selectivity, direct and cationic thiazole dyes were substituted in the alkaline Congo Red and the Phorwhite BBU procedure. With the former technic Diphenyl Brilliant Yellow 8G, Clayton Yellow, Thiazol Yellow, Thioflavine T and Seto Flavine T imparted strong to intense selective fluorescence to amyloid. Under the conditions of the Phorwhite BBU reaction these dyes were suitable only for formalin-fixed amyloid. Several thiazole dyes did not fluoresce. Fluorescence is a function of the molecular orbital system, the thiazole rings per se cannot induce fluorescence. Paper chromatograms indicated two or more fractions in the dyes studied. Different samples of the same dye can vary significantly in their staining and fluorescence properties. This heterogeneity is inherent in the mode of synthesis. In some cases the cationic thiazole dyes rendered certain amyloid deposits, e.g. in vessel walls, intensely fluorescent; other amyloid deposits in the same sections showed only weak fluorescence. Further studies are required to correlate these peculiar patterns with immunological data on amyloid types.

Mallory bodies: lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group.

Mallory's alcoholic hyalin in hepatocytes was found also in other diseases and is now referred to as Mallory bodies. Data concerning their histochemical, immuno and electron microscopic properties are partly contradictory. In this study, early stages of Mallory bodies reacted strongly with configurational technics for myosins; affinity tended to decrease when material with the properties of keratohyalin and the matrix of stratum corneum was formed. Thus, many Mallory bodies contained histochemically distinct myoid and keratin-like proteins. Electron microscopists demonstrated thick and thin filaments resembling contractile systems in Mallory bodies; the failure of immunologists to visualize actomyosin may be due to the heterogeneity of these proteins. The currently popular term prekeratin has been applied to a variety of substances extracted from epidermis, hoof and hair under different conditions. The prekeratin of recent immunofluorescence studies seems to contain mainly epidermin and low molecular matrix proteins; both were studied extensively by chemists. Epithelial filaments, including tonofibrils and contractile fibrils regarded as a subgroup of myofibrils, were well known half a century ago, but were banished by electron microscopy. Observations in this study and data on epidermal actomyosin indicate that different proteins of the k-m-e-f group can indeed coexist in epithelial cells. The formation and resolution of Mallory bodies can be regarded as an example of the well known shifts of epithelial cells between secretory and keratinizing states.

On Schiff's bases and aldehyde-fuchsin: a review from H. Schiff to R. D. Lillie.

In histochemistry aldehyde-fuchsin is widely regarded as an azomethine compound, though this hypothesis cannot explain the variety of reaction products. Infrared spectroscopy did not show a C = N bond. It was therefore deemed of interest to review chemical studies of aldehyde-fuchsin and other Schiff's bases by Schiff and his contemporaries. Schiff regarded reaction products of low molecular aliphatic aldehydes, e.g. acetaldehyde, and aromatic amines as diarylamines; aldehyde-fuchsin was assigned a 2:3 (dye:aldehyde) formula. These reactions were facilitated by alcohol and HCl. Others suggested condensation of two aldehyde molecules which carried a secondary and a tertiary amine respectively. Eibner proved that these compounds were ethylenes, not azomethines, and contained two secondary amines. Condensation of such bases produced ethylenic polymers, the Schultz's bases. Aromatic aldehydes readily yielded azomethines; aliphatic aldehydes formed --C = N-- bonds only during prolonged heating. These findings are in agreement with recent chemical data. Clearly, the term Schiff's bases is not synonymous with azomethines, but denotes any reaction product of aldehydes and amines. In 1962, Lillie's histochemical studies confirmed "the secondary amine nature of aldehyde aryl amine condensation". Thus, chemical and histochemical studies from Schiff in the 1860's to Lillie in the 1960's indicate that aldehyde-fuchsin is not an azomethine compound, but contains diarylamines and their derivatives.

On the mechanism of Mallory's phosphotungstic acid-haematoxylin stain.

The mechanism of Mallory's (1900) phosphotungstic acid-haematoxylin (PTAH) stain is not yet fully understood; staining properties vary with the age of the dye solution. In this study, a 1 year-old solution was employed as described previously. Paper chromatograms revealed blue, red and yellow components of phosphotungstic acid-haematoxylin. Modification of reactive groups as well as consecutive treatment of sections with haematoxylin and phosphotungstic acid indicated hydrogen bond formation by the blue component. Substitution of brazilin for haematoxylin proved that two free phenolic --OH groups per dye residue are essential for binding of the blue phosphotungstic acid-haematoxylin chelate. Uptake of the red fraction was determined by the polyacid moiety of the dye. Van der Waals forces presumably contributed to dye binding, but did not govern the selectivity of the red and blue components of PTAH for various tissue structures.

Histochemical investigations of different types of collagen.

During histochemical studies of connective tissue in the early 1960's, striking differences were observed between basement membranes and collagen fibers. A third histochemically distinct collagen was identified in premature infants. At that time, these findings could not be correlated with chemical data. However, during the last decade chemists described several types of collagen. Correlation of chemical, histochemical and immunofluorescence data indicated that the tendon-type collagen seen in coarse collagen fibers, e.g. in skin and adventitia of adults, contains type I collagen. The distribution of embryonic-type or pseudo-elastic collagen was similar to that of type III collagen; the major exception was reticulum fibers. Since antibodies and dyes are bound at different sites, it seems possible that fibers with the immunofluorescence characteristics of type III collagen may differ in their binding sites for other reagents. The trypsin-lresistant protein of basement membranes corresponded to type IV collagens. The collagen formed in glomerulosclerosis was histochemically indistinguishable from tendon-type, i.e. type I, collagen. A review or early literature since 1850 showed that histologists repeatedly described distinct chemical and histochemical differences between various collagens. These long ignored findings can easily be fitted into the framework of current collagen chemistry.

Infrared fluorescence microscopy of stained tissues: principles and technic.

Infrared photomicrography was used extensively from 1927 to the 1940's, but received little attention during the last decades. However, studies of infrared fluorescence of stained sections could not be found in the accessible literature. Ramsley (1968) published quantitative data on infrared fluorescence of approximately 250 dyes bound to textile fibers. The intensity of infrared fluorescence of many dyes varied widely with the substrate. It was therefore deemed of interest to determine whether or not similar differences in infrared fluorescence may occur when dyes are bound to histochemically distinct tissue structures. Myofibrils and collagens stained with triarylmethane dyes were chosen as test objects. Kodak infrared cut-off filter No. 301 and Wratten filter #16 were used as exciter filters to remove infrared and UV-blue and the light of a xenon lamp. Wratten filter #70 and #89B were employed as barrier filters. Infrared radiation was recorded with Kodak Ektachrome infrared film. To facilitate correlation of infrared fluorescence patterns with visible images, tissues were photographed also with conventional color film. Stained myofibrils, e.g. in myoepithelium, smooth and striated muscle emitted strong infrared fluorescence; collagen showed little or no fluorescence. Barrier filter Wratten #70 permitted simultaneous demonstration of infrared fluorescence and of non-fluorescent structures and thus facilitated histopathological studies. Preliminary findings indicate decrease or loss of infrared fluorescence of stained muscle fibers in various lesions, e.g. myocardial infarction, Duchenne-type muscular dystrophy.

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.

Aldehyde-fuchsin: historical and chemical considerations.

The staining mechanisms of Gomori's aldehyde-fuchsin are not yet fully understood. It seemed therefore timely to review the history of this dye class in context with current dye and aldehyde chemistry. In 1861 Lauth treated basic fuchsin with acetaldehyde. This dye became known as Aldehyde Blue, but consisted of violet and blue dyes. Schiff (1866) studied several aldehyde-fuchsins; these compounds contained two molecules of dye and three molecules of aldehyde. Acetaldehyde-fuchsin prepared according to Schiff's directions showed staining properties similar to those of Gomori's aldehyde-fuchsin. This dye class was soon superseded by new dyes more suitable for textile dyeing, and chemical investigations of aldehyde-fuchsins ceased around the turn of the century. Gomori's aldehyde-fuchsin has been regarded as a Schiff base. However, according to chemical data, low molecular aliphatic aldehydes and aromatic amines tend to form condensation products. Correlations of chemical and histochemical observations suggest such processes during aging of dye solutions. Models of dimers and polymers of aldehyde-fuchsin could be built without steric hindrance. The nature of the bonds formed by various components of aldehyde-fuchsin solutions is not clear. However, cystine in proteins, e.g. in basement membranes, apparently does not play a role in the binding of aldehyde-fuchsin by unoxidized Carnoy- or methacarn-fixed sections.

Demonstration of amyloid with Mesitol WLS-Congo Red: application of a textile auxiliary to histochemistry.

Previous histochemical investigations demonstrated similarities in the binding of Congo Red and other direct cotton dyes by amyloid and cellulose. It seemed therefore of interest to determine whether or not the cellulose-like reactivity of amyloid extends also to dye solutions containing an anionic reserving agent. These reagents are used in the dyeing of wool-cellulose (Halbwolle) fabrics to prevent binding of direct cotton dyes by proteins. Mesitol WLS-Congo Red solutions stained amyloid selectively; other tissue structures, except some hyaline deposits in arterioles, remained unstained. The cause of this non-specific reaction could not be determined with certainty. Therefore, the alkaline Congo Red method is recommended for histochemical identification of amyloid. However, the Mesitol WLS-Congo Red technic was very useful for demonstration of amyloid after prolonged storage of tissues in formalin; amyloid in such material showed little or no reactivity with the alkaline Congo Red or the Sirius dye methods. This pilot study indicates that anionic reserving agents can be effectively employed under conditions of histochemical technics.

Demonstration of phosphates in calcium deposits: a modification of von Kossa's reaction.

It has been suggested that in von Kóss'as technic silver cations replace calcium bound to phosphate or carbonate groups and are then reduced to black metallic silver during exposure to light. However, in test tube experiments silver phosphate retains its yellow color for days. These differences between reactions of pure calcium phosphates and calcium deposits in tissues were emphasized already by von Kóssa; he regarded only the initial yellow coloration of calcium diagnostic for calcium phosphates and deplored the subsequent blackening caused by organic compounds. Von Kóssa's experiments were easily reproducible. A review of the literature showed that reduction of silver nitrate by organic compounds was well known in the 19th century. For histochemical studies of phosphates it was deemed desirable to avoid the formation of black by-products. Sections of paraffin-embedded human tissues were exposed to solutions of silver nitrate in subdued light or darkness then treated with sodium thiosulfate. Silver phosphate was yellow to yellowish brown; other tissue structures remained colorless. No darkening was observed in sections stored for eight years. Other compounds which form yellow silver salts, e.g. iodides and periodates, are unlikely to occur in paraffin sections of human tissues.