Faye Sweat Waldrop

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.

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 combined PAS‐myofibril stain for demonstration of early lesions of striated muscle

To facilitate studies of early myocardial lesions a method was developed for simultaneous demonstration of saccharides and muscle proteins. Deparaffinized sections were treated consecutively with the PAS reaction, tannic acid, phosphomolybdic acid and Levanol fast cyanine 5RN. A and Z bands in normal muscle were stained blue. I bands were coloured yellow. Under various pathological conditions A and Z bands lost their affinity for Levanol fast cyanine 5RN, with and without appearance of PAS‐positive material in the Z bands.

Fluorescence microscopic distinction between elastin and collagen

SummaryDistinction between elastin and collagen in arteriosclerotic lesions is difficult because immature and incompletely cross-linked collagen bind so-called elastica stains; furthermore, abnormal collagen can lack cross-striation and thus resemble elastin in electron microscopy. However, collagen and elastin differ significantly in their content of basic amino acids and hence in their affinity for heteropolyacids. This chemical difference was utilized for the development of a fluorescence microscopic method for distinction between collagen and elastin.Paraffin sections of human autopsy material were treated with a 1% aqueous solution of phosphomolybdic acid (PMA) for five minutes, rinsed in distilled water, dehydrated and mounted. Other series were treated with the PMA-molybdenum blue reaction and with various special stains.Only elastic membranes of aorta, the elastica interna and externa of sizable arteries, and “true” elastic fibers remained strongly fluorescent; the autofluorescence of collagen, reticulum fibers, basement membranes, pseudo-elastic fibers, and “elastic” membranes in small arteries was quenched. In other series PMA abolished the fluorescence of basic fluorochromes.Correlation of fluorescence and direct light microscopic observations with chemical and electron microscopic data showed that the PMA-fluorescence method permits distinction between elastin and various types of collagen.

Myoid fibrils in epithelial cells: Studies of intestine, biliary and pancreatic pathways, trachea, bronchi, and testis

SummaryCytoplasmic filaments have been studied extensively by electron microscopy, but the histochemical nature of such fibrils in non-keratinizing epithelia has not been systematically investigated. During studies of early arterial lesions we observed structures with the staining properties of myosins in epithelial cells of various organs. The configurational staining, polarization and fluorescence microscopic properties of these myoid structures were compared with those of myofibrils in smooth muscle and classical myoepithelial cells. The following structures showed the characteristics of myofibrils: the terminal web in columnar epithelial cells of intestine, trachea, bronchi, bile ducts, pancreatic ducts and ductus epididymidis, the pericanalicular layer of bile and pancreatic canaliculi, fibers in the caudal tube of spermatids and the flagella of spermatozoa. Cilia, e.g. of respiratory epithelium, tonofibrils in squamous epithelium and nerve axons did not react.These studies indicate significant histochemical differences between cytoplasmic filaments. Different types of intracellular fibrils can be found in the same cell, e.g. in respiratory epithelium.

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

SummaryVerhoeff (1908) recommended an iron-hematein formula containing Lugols solution for demonstration of elastic tissue; sections are differentiated until desired staining patterns are obtained. Verhoeffs stain colored a variety of tissue structures and showed higher substantivity for myelin sheaths than for elastin. Addition of HCL or omission of Lugols 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 Lugols 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.Verhoeffs 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, Verhoeffs 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 Verhoeffs elastica stain as a method for myelin sheaths.

Aldehyde-fuchsin: Historical and chemical considerations

SummaryThe staining mechanisms of Gomoris 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 Schiffs directions showed staining properties similar to those of Gomoris 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. Gomoris 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.