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H Puchtler

Publications and source records attributed to H Puchtler.

At least 19 recordsLinked to original sources
PubMed1988

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.

Azo Compounds↗
PubMed1986

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.

Aorta↗
PubMed1985

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)

Acids↗
PubMed1985

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.

Fixatives↗
PubMed1985

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)

Acetates↗
PubMed1985

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.

Amyloid↗
PubMed1979

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.

Aldehydes↗
PubMed1979

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.

Adolescent↗
PubMed1979

Histochemical observations on Pneumocystis carinii: selective demonstration of honeycomb forms.

Histochemical investigations of pulmonary lesions indicated selective coloration of membranes of honeycomb stages of Pneumocystis carinii by the periodic acid--sodium bisulfite--resorcin-fuchsin reaction for basement membranes; mucus, fibrin and other deposits in respiratory pathways did not react. These membranes were colored selectively also by the picro-Sirius Red F3BA method for collagens; fungi in tissues from patients with candidiasis remained unstained. For simultaneous demonstration of honeycomb and cyst forms of Pneumocystis carinii, sections were prestained with Grocott's modification of Gomori's methenamine-silver nitrate technic and then treated with the periodic acid-Schiff (PAS) or picro-Sirius Red F3BA reaction. In contrast to other Gram-positive microorganisms, cysts of Pneumocystis carinii were immediately decolorized by acetone-ether mixtures; this indicates differences in the mode of dye binding. Frequently, only one stage of Pneumocystis carinii was found in a given area. Hence a combination of reactions showing different stages is recommended for studies of small tissue samples.

Basement Membrane↗
PubMed1979

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

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.

Aorta↗
PubMed1978

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.

Amyloid↗
PubMed1978

Silver impregnation methods for reticulum fibers and reticulin: a re-investigation of their origins and specificity.

Maresch (1905) introduced Bielschowsky's silver impregnation technic for neurofibrils as a stain for reticulum fibers, but emphasized the nonspecificity of such procedures. This lack of specificity has been confirmed repeatedly. Yet, since the 1920's the definition of "reticulin" and studies of its distribution were based solely on silver impregnation technics. The chemical mechanism and specificity of this group of stains is obscure. Application of Gomori's and Wilder's methods to human tissues showed variations of staining patterns with the fixatives and technics employed. Besides reticulum fibers, various other tissue structures, e.g. I bands of striated muscle, fibers in nervous tissues, and model substances, e.g. polysaccharides, egg white, gliadin, were also stained. Deposition of silver compounds on reticulum fibers was limited to an easily removable substance; the remaining collagen component did not bind silver. These histochemical studies indicate that silver impregnation technics for reticulum fibers have no chemical significance and cannot be considered as histochemical technics for "reticulin" or type III collagen.

Connective Tissue Cells↗
PubMed1978

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.

Aorta↗
PubMed1976

Light microscopic distinction between elastin, pseudo-elastica (type III collagen?) AND INTERSTITIAL COLLAGEN.

Distinction between elastin and collagen in arteriosclerotic lesions is difficult because the so-called elastica stains are bound also by collagen fibers which resemble collagen of premature infants. Investigations of effects of organic solvents on dye binding led to the development of methods for selective demonstration of pseudo-elastica, and for simultaneous visualization of elastin and pseudo-elastica in contrasting colors. Paraffin sections of human autopsy material were stained with solutions of resorcin-fuchsin, orcein or aldehyde fuchsin in absolute ethanol. In other series, sections pretreated with this resorcin-fuchsin solution were counter-stained with tannic acid-phosphomolybdic acid (TP)-dye technics. Solutions of these "elastica stains" in absolute ethanol colored only pseudo-elastica; elastin, e.g. elastic membranes of aorta, remained unstained. In sections counterstained with TP-dye technics elastin was colored red; pseudo-elastica retained the purplish blue coloration imparted by resorcin-fuchsin. Other collagens were stained yellow. A review of the literature showed that until the 1920's elastin was classified as a gelatinoid of the collagen group. Elastic fibers were identified by mechanical properties, not a particular chemical composition. Hence, the elastic fibers of classical histology cannot be equated with the elastin of modern chemistry. Correlation of histochemical observations with chemical data indicates that the collagenous pseudo-elastica corresponds to [alpha1(III)]3 collagen.

Adult↗
PubMed1976

Light microscopic demonstration of myoid material in nuclei.

During the development of configurational staining methods for proteins of the myosin-fibrin group, nuclei showed staining properties similar to those of myofibrils. This dye binding could be attributed to nuclear alpha-helical proteins. More recent chemical and electron microscopic studies demonstrated actomyosins in nuclei of various species. Possible roles of nuclear actomyosin in chromosome movements and condensation and in cell proliferation have been suggested. It seems therefore permissible to assume that the tannic acid-phosphomolybdic acid (TP)-Levanol Fast Cyanine 5RN method and similar technics visualize myosin in nuclei. Comparative studies of actomyosins from various sites indicated significant chemical an histochemical differences. It is therefore suggested that, in analogy to the different classes of collagens, there may be several subgroups of myosin which differ in their physico-chemical properties and sensitivity to fixation procedures and pathological conditions.

Actomyosin↗