Holde Puchtler

ON THE BINDING OF CONGO RED BY AMYLOID

Congo red in alkaline 80% ethyl alcohol solution saturated with NaCl stains amyloid selectively. The distribution of the dye is very similar to that observed in serial sections stained with Highmans Congo red method. In sections stained with Congo red in aqueous or alcoholic solutions without differentiation all tissue structures are intensely colored, but only amyloid is dichroic. The rapid removal of dye by alkaline solutions from structures other than amyloid suggests ionic or salt-type bonds between Congo red and these structures. In contrast, the binding of Congo red by amyloid from alkaline alcoholic solutions and the increase in intensity of staining upon addition of NaCl indicate a non-ionic type linkage between amyloid and dye. The effects of deamination, acetylation and various oxidation procedures on the binding of Congo red indicate interaction of the dye with hydroxyl groups of amyloid. According to data on the direct dyeing of cellulose in textile dyeing, Congo red is adsorbed by hydrogen bonding between hydroxyl groups of the polysaccharide chains and the amino groups of the dye. It seems probable that a similar mechanism is involved in the binding of Congo red by amyloid. It is suggested that the selective staining of amyloid with Congo red is due to steric configurations of amyloid or one of its components which favor non-ionic binding of the eye.

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

SummaryFormalin 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.

Polarization microscopic studies of connective tissue stained with picro-sirius red FBA.

Summary Polarization microscopic studies are usually carried out on unstained tissues. However, polarization microscopy of dyed fibers has long been used in the textile industry and observations were correlated with x-ray diffraction data. In this study the principles of polarization microscopy of stained fibers were applied to connective tissue. Human autopsy material was fixed in buffered and unbuffered formalin, Zenkerformol, Carnoys fluid or methacarn. Deparaffinized sections were stained in a 0.1% solution of Sirius F3BA in saturated aqueous picric acid for 30 minutes, dehydrated and mounted. Sirius red F3BA enhanced the birefringence of collagen and reticulum fibers significantly. Elastin, basement membranes, ring fibers, and related structures were isotropic. Collagen fibers in various lesions, e. g., glomerular fibrosis, early arteriosclerosis, were easily identifiable by their strong birefringence. The stain was found advantageous for general pathology because it permits direct comparison of familiar staining patterns with the polarization microscopic pitcure. Correlation of polarization microscopic observations with electron microscopic and x-ray diffraction data demonstrated relations between molecular orientation of connective tissue structures and birefringence.

Methacarn (methanol-Carnoy) fixation

SummaryAccording to chemical data, methanol raises the shrinkage temperature of collagen significantly more than ethanol (86° C versus 70° C). Since increase of shrinkage temperature appears desirable in tissues to be embedded in paraffin, methanol was substituted for ethanol in Carnoys fluid. This methanol-Carnoy mixture is referred to as methacarn solution. The fixation-embedding procedure was similar to that described in the study of Carnoy fixation. Methacarn-fixed sections showed little or no shrinkage and compared well with material fixed in Carnoys or Zenkers fluid. Myofibrils, especially in endothelial and epithelial cells, were more prominent in methacarn- than in Carnoy-fixed tissues.A review of the chemical literature showed that methanol, ethanol and chloroform stabilize or even enhance helical conformations of proteins, presumably by strengthening of hydrogen bonds. Interference with hydrophobic bonds causes unfolding and/or structural rearrangements in globular proteins. The twin-helical structure of DNA collapses in alcoholic solutions. Hence, methacarn fixation can be expected to preserve the helical proteins in myofibrils and collagen, but the conformations of globular proteins and DNA will be significantly altered. Literature on conformational effects produced by fixatives used in electron microscopy was also reviewed. Glutaraldehyde and OsO4 cause considerable loss of helix (22–29% and 39–66% respectively). KMnO4 and glutaraldehyde followed by OsO4 produce extensive transitions from helical to random-coil conformations similar to those seen in powerful denaturants such as 8 M urea. Evidently these fixatives are unsuitable for studies of helical proteins. In contrast ethylene glycol preserves helical conformations.

Investigation of staining, polarization and fluorescence-microscopic properties of myoendothelial cells

Electron microscopists have described in vascular endothelial cells filaments which resemble myofibrils. These fibrils cannot be demonstrated with conventional staining techniques. On the basis of previous investigations of relations between dye structure and affinity for muscle fibres, a staining method for demonstration of myoendothelial cells by direct, polarization and fluorescence microscopy has been developed. Carnoy‐fixed paraffin sections were treated consecutively with Kernechtrot, tannic and phosphomolybdic acid and counter‐stained with Levanol fast cyanine 5RN. This procedure stained myoendothelial cells and muscle fibres deep blue, connective tissues yellow and nuclei pink. For polarization and fluorescence‐microscopic studies thiazine red R was substituted for Levanol fast cyanine 5RN and Kernechtrot was replaced by Mayers acid hæmalum. The light‐microscopic characteristics of fibrils in endothelial cells and in smooth muscle cells were identical.

Histochemical specifity of staining methods for connective tissue fibers: Resorcin-fuchsin and van Gieson's picro-fuchsin

Conclusions and summaryDepending on the pretreatment of tissue sections, resorcin-fuchsin stained collagen, reticulum fibers and/or basement membranes and ring fibers intensely. It must therefore be concluded that resorcin-fuchsin is not specific for the protein elastin, that is elastic fibers in the chemical sense of the term.Studies of van Gieson-type stains showed relation between dye structure and affinity for connective tissue fibers. These observations are in good agreement with data from textile and leather dyeing that the behaviour of sulfonated dyes is largely determined by the configuration of the dye molecule and the presence of additional reactive groups.Polarization microscopic studies of stained sections — based on data derived from textile chemistry — demonstrated the possibility to obtain information concerning the submicroscopic structure of tissue components. Several methods, though still in the experimental stage, have been found valuable for the study of pathological lesions of connective tissue. Because electron microscopy is unsuitable for routine histopathology, it is highly desirable to develop convenient methods for the study of submicroscopic structures in general hospital pathology.

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

SummaryCurrent chemical concepts were applied to Weigerts, M. Heidenhains and Verhoeffs iron hemateins, Mayers 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 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.

Carnoy fixation: practical and theoretical considerations.

SummaryThe Carnoy-fixation, paraffin-embedding procedure was modified to minimize shrinkage and to suit the schedule of general histology laboratories. Blocks of tissues were fixed in Carnoys fluid overnight, transferred to absolute alcohol in the morning and washed in three changes of alcohol for a total of 6–8 hours. Tissues were then left in methyl benzoate overnight, transferred to an Autotechnicon in the morning, cleared in xylene and xylene-paraffin 1 hour each and infiltrated in 2–3 changes of paraffin for a total of 4–5 hours. This procedure has worked well in our hands for nine years.A review of the chemical literature showed that the components of Carnoys fluid-ethanol, chloroform and acetic acid-can interact by hydrogen bond formation with each other and with various groups in tissues. These association compounds apparently stabilize tissue structures and prevent or minimize shrinkage. Ethanol alone causes collapse of protein structures; exposure must be limited to eight hours or less. Addition of water to an ethanol-acetic acid solution causes considerable swelling of proteins and subsequent shrinkage in absolute alcohol; hence, the ratio fixative: tissue should be not less than 20∶1. In our experience, Carnoys fluid is the fixative of choice for studies of fibrous proteins and associated carbohydrates by histochemical and special staining technics.

Silver impregnation methods for reticulum fibers and reticulin: A re-investigation of their origins and specifity

SummaryMaresch (1905) introduced Bielschowskys silver impregnation technic for neurofibrils as a stain for reticulum fibers, but emphasized the nonspecifity of such procedures. This lack of specifity has been confirmed repeatedly. Yet, since the 1920s the definition of “reticulin” and studies of its distribution were based solely on silver impregnation technics. The chemical mechanism and specifity of this group of stains is obscure. Application of Gomoris and Wilders 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.

A review of early concepts of amyloid in context with contemporary chemical literature from 1839 to 1859.

Early concepts of amyloid are reviewed in context with chemical literature from 1839-1859. Cellulose was first described in 1839. Treatment with sulfuric acid converted cellulose into a substance which was colored blue by iodine. This cellulose derivative was called amyloid. Virchow applied the chemical tests for starch and cellulose to human tissues. The substance in waxy degeneration reacted like immature cellulose. Virchow did not coin the term amyloid for this substance but suggested it as a compromise. Virchow restricted the term amyloid to the carbohydrate moiety and explicitly excluded the protein fraction which he had described earlier. Friedreich and Kekulé (1859) studied an evidently impure sample of amyloid and concluded that it did not contain polysaccharides. Since then the protein moiety has been studied extensively, but the carbohydrate fraction has been largely neglected. It appears possible that investigations of the carbohydrates at sites of amyloid formation would aid the understanding of the pathogenesis of amyloidosis2.