bluuebunny/crossref_metadata_2025 huggingface dataset:
IMPROVED STAINING FOR PEROXIDASE WITH BENZIDINE AND IMPROVED DOUBLE STAINING IMMUNOPEROXIDASE PROCEDURES
Authors: STRAUS WERNER
Published: April 2011

The sensitivity of the cytochemical reaction for peroxidase with benzidine and H2O2 could be much enhanced and a noncrystalline, blue-brown reaction product could be obtained by decreasing the concentration of ethanol (for dissolving benzidine) and by increasing the time and temperature of incubation. This method, together with a new method for the inactivation of residual (injected) peroxidase, were incorporated in double staining procedures for horseradish peroxidase (HRP) and its antibody (antigen-antibody complexes) and in double staining procedures for the antibody to HRP and acid phosphatase activity. Double staining in contrasting colors was also applied to detect rabbit antibodies against two antigens (HRP and rat anti-HRP γ-globulin) in the same section of popliteal lymph nodes. It was found that the antibody against each antigen appeared in different plasma cells whether the rabbits were immunized against the two antigens separately or against both antigens together as antigen-antibody complexes. Certain technical problems arising in double staining procedures are discussed.
LOSS OF PROTEINPOLYSACCHARIDES AT SITES WHERE BONE MINERALIZATION IS INITIATED
Authors: BAYLINK D. , WERGEDAL J. , THOMPSON E.
Published: April 2011

In both ground sections and demineralized frozen sections of the rat tibial cortex, osteoid but not mature bone matrix stained for proteinpolysaccharides with the Alcian Blue and toluidine blue techniques. The loss of proteinpolysaccharide staining occurred precisely at the mineralizing front, which was identified by in vivo lead or procion markers, not only in normal animals but also in animals in which osteoid width was either increasing or decreasing. In vitro, both proteases and saccharidases abolished proteinpolysaccharide staining of osteoid. Critical electrolyte concentration and other procedures indicated that the major acid polysaccharide component in osteoid is chondroitin sulfate. Consistent with these findings, electron microprobe analyses revealed that sulfur concentration was high in osteoid but dropped abruptly as calcium concentration increased at the mineralizing front. The precise synchronization between loss of proteinpolysaccharides and onset of mineralization under various experimental conditions provides strong indirect evidence that the loss of these macromolecules is somehow involved in initiation of mineralization in bone.
A CRITICAL EVALUATION OF THE HISTOCHEMICAL METHODS FOR CARBONIC ANHYDRASE
Authors: MUTHER THOMAS F.
Published: April 2011

The histochemical methods for carbonic anhydrase are not based on the postulated dehydration of HCO3–. The staining is caused by the formation of an unknown reactive Co compound in the surface layer secondary to enzyme-independent alkalinization of the medium. Kinetic analysis of the reaction shows that loss of CO2 from the medium is rate-limiting. Carbonic anhydrase inhibitors delay the staining by interacting with Co and not by inhibiting the enzyme; they are effective when used after the reaction is complete. The reaction can also be inhibited by agents which are not carbonic anhydrase inhibitors, such as sodium lauryl sulfate and 5-aminothiadiazole, but not by in vivo administered acetazolamide. A comparison of the effect of various fixatives on the biochemical and histochemical enzyme activity shows no correlation. While carbonic anhydrase itself is stained by the reaction, the methods lack the claimed specificity for it.
l-HOMOARGININE, A SPECIFIC INHIBITOR OF LIVER-TYPE ALKALINE PHOSPHATASE, APPLIED TO THE RECOGNITION OF LIVER-TYPE ENZYME ACTIVITY IN RAT INTESTINE
Authors: RUFO MYRA BLUESTEIN , FISHMAN WILLIAM H.
Published: April 2011

l-Homoarginine, an inhibitor of alkaline phosphatase, has been applied for the study of the localization of alkaline phosphatase activity in rat liver and intestine. Five substrates were employed: Na α-naphthyl acid phosphate, naphthol AS-BI phosphate, Na β-glycerophosphate, o-carboxyphenylphosphate and O-ethanolamine phosphate. Diazonium coupling was employed with the naphthol substrates; the Gomori calcium-cobalt method was used with the others. Each substrate was also employed in the presence of 12.5 mMl-phenylalanine, an intestinal (brush border-specific) alkaline phosphatase inhibitor. The liver-type enzyme, in both the liver parenchyma and the lamina propria of the small intestine, was sensitive to 12.5 mMl-homoarginine in varying degrees, depending on the substrate used. With Na α-naphthyl acid phosphate, a total inhibition was observed histochemically; l-homoarginine inhibited partially purified liver alkaline phosphatase by 97%, as measured biochemically. The alkaline phosphatase of leukocytes and macrophages of the intestinal lamina propria was inhibited in the presence of l-homoarginine but not of l-phenylalanine. Reactive elements that resembled the macrophages of the lamina propria were present in the liver. The activity in these cells was also inhibited by l-homoarginine.
IMMUNOCYTOCHEMICAL LOCALIZATION OF THE EXTRACELLULAR POLYSACCHARIDE ALGINIC ACID IN THE BROWN SEAWEED, FUCUS DISTICHUS
Authors: VREELAND VALERIE
Published: April 2011

Fluorescent and peroxidase-labeled antibody techniques were employed to localize alginate, which is one component in the complex extracellular acidic polysaccharide situation of the brown seaweed, Fucus distichus. The alginate antigen was extracted and characterized by a variety of methods. It had a low degree of polymerization and a mannuronic acid to guluronic acid ratio of 2.2. No contaminants were detected. The specificity of rabbit antisera was tested by immunodiffusion. Antisera reacted with alginate and fractions of alginate but not with other brown algal polysaccharides. When cryostat sections of Fucus proved unsuitable, 1-µ sections embedded in glycol methacrylate were used for indirect fluorescent or peroxidase antibody staining. Highly sulfated extracellular polysaccharides and intracellular phenolic compounds were implicated in nonspecific staining. Texture artifacts were caused by solubility of alginates and other polysaccharides from embedded sections. Alginate was localized in variable patterns in all cell walls and some matrix regions. The applicability of the methods is discussed.
ELECTRON PROBE ANALYSIS OF THE CALCIUM DISTRIBUTION IN CELLS OF THE EMBRYONIC CHICK CHORIOALLANTOIC MEMBRANE I. A CRITICAL EVALUATION OF TECHNIQUES
Authors: COLEMAN JAMES R. , TEREPKA A. RAYMOND
Published: April 2011

The chorioallantoic membrane of the developing chick embryo is an epithelium that actively transports calcium. The methodology utilized to prepare this soft tissue for calcium localization with the electron probe x-ray microanalyzer is presented in detail. The preparative procedures are evaluated according to general histochemical principles and in relationship specifically to electron probe investigations. It is shown that the method employed in these studies preserves the normal fine structure of the tissue, prevents selective loss of calcium, permits only minor losses of total calcium and appears to maintain the distribution of calcium that existed in vivo. Examples are presented of artifacts that can be induced during tissue sectioning and mounting procedures. Problems of defining electron probe resolution in biologic specimens are discussed, and the critical importance of evaluating x-ray images in association with simultaneously generated sample current images is emphasized.
FLUORESCENCE HISTOCHEMISTRY OF BIOGENIC MONOAMINES A STUDY OF THE CAPACITY OF VARIOUS CARBONYL COMPOUNDS TO FORM FLUOROPHORES WITH BIOGENIC MONOAMINES IN GAS PHASE REACTIONS
Authors: AXELSSON STURE , BJÖRKLUND ANDERS , LINDVALL OLLE
Published: April 2011

The capacity of biogenic amines to form fluorophores in histochemical gas phase reactions has been tested with 20 compounds having a carbonyl group (> C = O) as a common characteristic. Significant visible fluorescence was induced from catecholamines and indolamines with aldehydes, ketones, α-keto acids and carboxylic acids, suggesting that all of these compounds can enter fluorophore-forming cyclization reactions under the histochemical gas phase conditions. The most "reactive" reagents are found among the low molecular aldehydes, and formaldehyde and glyoxylic acid seem to be the most suitable reagents, combining high reactivity with good selectivity. Fluorescence, interesting for the histochemical demonstration of N-acetylated indolamines, was obtained from melatonin and N-acetyl-5-hydroxytryptamine with some acid reagents (glyoxylic acid, formic acid, acetic acid and pyruvic acid). A direct cyclodehydration according to the classical Bischler-Napieralski reaction is the most likely mechanism underlying this fluorophore formation. The usefulness of the various carbonyl reagents for the fluorescence histochemical demonstration of other biologically interesting amines, such as histamine, p-tyramine and octopamine, has been specially investigated.
THE EFFECT OF LIGHT MICROSCOPIC FIXATIVES ON THE RETENTION OF GLYCOGEN IN PROTEIN MATRICES AND THE PARTICULATE STATE OF NATIVE GLYCOGEN
Authors: SMITHERMAN M. L. , LAZAROW A. , SORENSON R. L.
Published: April 2011

The percentage of glycogen fixed or retained (not extracted with water) in rat liver slices and in model systems by various fixatives has been studied. After homogenization of the fixed liver slices, more glycogen was retained in the residue of liver initially fixed with Rossman's fixative (70%) than with Bouin's fixative (50%); little glycogen was retained following fixation with 3.7% formaldehyde (12%). Macromolecular glycogen extracted from rat liver and exposed for 2-16 hr at 3°C to various fixatives was found to form a stable solution when resuspended in water. After an exposure to Rossman's fixative (pH 1.7), components of Rossman's fixative and 3.7% formaldehyde (pH 6), the resuspended glycogen retained its macromolecular state; an exposure to Bouin's fixative (pH 1.9) dispersed the glycogen into smaller particles similar to that observed following treatment with 0.1 N HCl (pH 1.5). Both the action of picric acid on proteins and the maintenance of the macromolecular state of glycogen during Rossman's fixation appear to facilitate the effective fixation of glycogen. A possible mechanism for the fixation of glycogen is discussed.
MICROSOMAL AND LYSOSOMAL ACID PHOSPHATASE ISOENZYMES OF MOUSE KIDNEY CHARACTERIZATION AND SEPARATION
Authors: LIN CHI-WEI , FISHMAN WILLIAM H.
Published: April 2011

Biochemical methods have been employed to characterize and separate acid phosphatases from lysosomal and microsomal fractions in order to decide whether different isoenzymes reside in these subcellular locations. Microsomal and lysosomal fractions of mouse kidney homogenate were isolated by differential centrifugation. Acid phosphatase of lysosomal fraction goes into solution after lysosomes have been repeatedly frozen and thawed, whereas acid phosphatase of microsomal fraction is firmly bound to the membrane and is freed of contamination by lysosomal enzyme after ultrasonication and centrifugation. The membrane-bound microsomal acid phosphatase is labile at 37°C, pH 4.9, more active toward phenolic substrates (phenyl phosphate and p-nitrophenyl phosphate) than β-glycerophosphate, α-naphthol phosphate or naphthol AS-BI phosphate. It also has a higher pH optimum (6.3), is resistant to l-tartrate and oxalate inhibition and has a slower electrophoretic migration rate in Triton X-100-impregnated polyacrylamide gels. The free lysosomal acid phosphatase is relatively heat-stable, is less active against phenolic substrates, is sensitive to l-tartrate and oxalate inhibition, has a lower pH optimum (5.6) and has a faster migration rate in electrophoresis. These two acid phosphatases can also be separated by diethylaminoethyl-cellulose chromatography. This study thus demonstrated the existence of an acid phosphatase isoenzyme in the microsomal membrane with different biochemical properties from the lysosomal isoenzyme of acid phosphatase.
ELEMENTAL ANALYSIS OF PRECIPITATES FORMED IN NUCLEI BY ANTIMONATE-OSMIUM TETROXIDE FIXATION
Authors: SPICER S. S. , SWANSON A. A.
Published: April 2011

Elements retained in cervical lymph nodes, isolated hepatic nuclei and salt-impregnated gels by fixation with antimonate- or pyrophosphate-containing and other osmium tetroxide solutions were assayed by nuclear activation analysis or by atomic absorption spectrophotometry. Salts preserved by the antimonate-osmium tetroxide fixative in lymph nodes, isolated nuclei and a KCl-enriched gel consisted almost entirely of potassium antimonate. The K+ in the precipitates in these specimens appeared to derive partially from that in the fixative solution and partially from that in the specimen. Salts preserved by the antimonate-osmium tetroxide fixative in an NaCl-supplemented gel consisted partly of potassium antimonate derived from the fixative as in unsupplemented gels and partly of sodium antimonate. The Na+ precipitated in this gel amounted to less than one-half that originally present. In comparison the pyrophosphate-osmium tetroxide solution retained higher levels of K+ in lymph nodes, nuclei and the KCl gel, but the potassium pyrophosphate was not evident as electron-opaque precipitates. The latter fixative was less effective in preserving Na+ in the NaCl gel. The pyrophosphate-containing fixative, which was about twice as efficient as the antimonate-containing solution in retaining the divalent cations, preserved 70% of the Mg++ and 100% of the Ca++ added to gels.
THE HISTOCHEMICAL DEMONSTRATION OF POLYADENYLIC ACID HYDROLASES IN RAT LIVER DURING AZO DYE CARCINOGENESIS
Authors: DAOUST R.
Published: April 2011

Films of polyadenylic acid (poly-A) were exposed to liver sections from 4-dimethylaminoazobenzene (DAB)-fed rats in order to determine whether the nucleases acting on these films, like the ribonucleases (RNases), are depressed during carcinogenesis. Normal liver parenchyma gave a positive reaction which was particularly intense in periportal areas. Livers from animals fed the basal control diet showed a similar distribution of enzyme activity but were generally more active than normal livers. In DAB-fed rats, the nodules of hepatic tissue gave intense reactions while the trabeculae of bile ducts and connective tissue, as well as the necrotic areas, were negative. The formation of hyperbasophilic foci at later stages of DAB feeding was accompanied by a loss of enzyme activity. The hepatomas, which apparently derived from such foci, showed weak or negligible activity. Thus the changes in RNases and poly-A hydrolases occur at different stages of the carcinogenic process. The loss of RNase activity precedes the neoplastic transformation while the decrease in the activity of poly-A hydrolases is closely associated with tumor formation, but the induced tumors are deficient in both types of nuclease activity.
A TECHNIQUE FOR THE PREPARATION OF MONOLAYERS OF MESOTHELIUM
Authors: CHENG HAZEL , BERRY MARGARET
Published: April 2011

A technique for the preparation of monolayers of mesothelial cells using gelatincoated slides is described. These preparations were adequate for both general histologic and radioautographic studies.
QUANTITATIVE ELECTRON PROBE ANALYSIS OF SOFT BIOLOGIC TISSUE FOR ELECTROLYTES
Authors: INGRAM F. DUANE , INGRAM MARY JO , HOGBEN C. ADRIAN M.
Published: April 2011

Electron probe analysis of soft biologic tissue requires the use of a tissue preparation that (a) presents a flat surface of the specimen to an electron beam in a vacuum, (b) can withstand the rigors of a vacuum, (c) can withstand damage by an electron beam and (d) causes no redistribution of the elements under study. A tissue preparation which also does not involve the use of aqueous solutions for any step of the preparation has been used for the examples of electron probe analyses presented here. Tissue is prepared by quick freezing at about –196°C, drying in a vacuum at between –60°C and –85°C, fixing with osmium tetroxide vapor and embedding in a low Cl epoxy. Calibration of the electron probe signals is accomplished with prepared solutions of 20% albumin and known quantities of electrolytes. Cell morphology is well preserved to the submicron level, but counting rates and signal to background ratios for the x-ray signals are low. With a 10-kV, 50-na electron beam, the spatial resolution is comparable to that of light microscopy with detection limits for K, Cl and Na of a few milliequivalents per liter. Analysis of frog skeletal muscle fibers yielded concentrations of 122.4 ± 2.8 (s.e.) mEq/liter for K, 10.5 ± 0.7 mEq/liter for Cl and 9.2 ± 1.0 mEq/liter for Na as the averages of nine fibers from one muscle. The Ca content in a discontinuous extracellular layer in frog skin was found to be as high as 4-5 M with a Ca:P ratio of an apatite when referenced to a natural fluoroapatite crystal.
CHEMICAL CHARACTERIZATION OF ASBESTOS BODY CORES BY ELECTRON MICROPROBE ANALYSIS
Authors: LANGER ARTHUR M. , RUBIN IVAN B. , SELIKOFF IRVING J.
Published: April 2011

Inhalation of asbestos may be associated with increased risk of developing malignant neoplasms. Some of the fibers become coated in the lung, resulting in "asbestos bodies." The occurrence of structures with the appearance of asbestos bodies in the lungs of urban dwellers the world over, individuals with no known exposure to these mineral fibers, has raised the question of whether the community at large may also have increased risk of neoplasia as the result of chance environmental asbestos exposure. Since other fibrous materials may also sometimes become so coated, epidemiology evaluation of the presence of asbestos bodies has been hampered by difficulties in obtaining absolute identification of the cores of the bodies found. Five fibrous silicates, consisting of four amphiboles (amosite, anthophyllite, crocidolite and tremolite) and one serpentine (chrysotile), constitute the asbestos mineral group. Chemically, they are diverse enough for unique identification. The electron microprobe analyzer permits microchemical analysis of particles in the sublight microscopic size range. Analysis of asbestos body cores requires particle selection, extraction from tissue matrix, a suitable conducting substrate, proper coating material, selection of optimal instrumental operating conditions and comparison of unknown cores with known fiber standards. In this investigation, asbestos body cores have been analyzed from tissues obtained from occupationally exposed individuals (known fiber exposure), laboratory animals (known exposure) and individuals with no known occupational exposure. Cores of bodies have been analyzed as amosite, chrysotile, chemically degraded chrysotile and cores of undetermined nature. Amosite fibers as cores of asbestos bodies show no marked chemical degradation even after prolonged biologic residence, whereas chrysotile asbestos cores are markedly degraded. Cores of asbestos bodies from the general population, from individuals with no known exposure, may consist of degraded chrysotile, synthetic silicate fibers and, in some cases, amphibole asbestos.
A DIRECT CYTOCHEMICAL METHOD FOR THE IDENTIFICATION OF LYSOZYME IN VARIOUS TISSUES
Authors: SCHOLNIK AARON P. , KASS LAWRENCE
Published: April 2011

This report describes a cytochemical technique for the demonstration of lysozyme in a variety of cells, including normal human and rabbit neutrophil granules, rabbit lacrimal gland cells and blasts obtained from bone marrow and peripheral blood of patients with myelomonocytic and histiomonocytic leukemia. It utilizes the dis-azo dye Biebrich scarlet which stains lysozyme as well as other basic proteins. The method is made specific for lysozyme by the use of N-acetylglucosamine oligosaccharides obtained from an acid hydrolysate of chitin. These greatly diminish the color reaction obtained when Biebrich scarlet is added to lysozyme, presumably by competing with Biebrich scarlet for the active site of the lysozyme molecule. Cytochemically, this is expressed as a marked diminution in the ability of Biebrich scarlet to stain the cytoplasm of lysozyme-containing cells if these cells have been pretreated with a hydrolysate of chitin. Evidence is presented that this stain is specific for lysozyme rather than a nonspecific reaction between Biebrich scarlet and tissue bases, particularly arginine.
EFFECTS OF ADRENALINE AND NORADRENALINE DISCHARGING AGENTS ON THE DIAZOSULFANILIC ACID AZURE A STAINING OF RAT ADRENAL MEDULLA
Authors: CATALANO R. A. , LILLIE R. D.
Published: April 2011

In normal glutaraldehyde-fixed rat adrenal medulla, islets of cells will stain brown with potassium iodate while the entire medulla will stain brown with potassium bichromate. The diazosulfanilic acid, pH 1 azure A sequence (DAS-AzA) stain revealed dark blue cytoplasmic staining of noradrenaline islet cells with the surrounding cytoplasmic areas a pale blue-green to almost colorless and the nuclei a moderate green. Two noradrenaline discharging agents, nicotine and reserpine, weakened or prevented all three staining reactions in these islets. Insulin injections, known to mobilize adrenaline from adrenaline-producing cells in the adrenal medulla did not impair the iodate or DAS-AzA reaction. These findings on glutaraldehyde-fixed adrenal medulla confirm the selectivity of the DAS-AzA reaction for noradrenaline cells in the adrenal medulla.
SOLUBLE HEMIN COMPOUNDS AS ULTRASTRUCTURAL TRACERS
Authors: ARONSON J. F. , FISHMAN A. P. , PIETRA G. G.
Published: April 2011

We have demonstrated that soluble hemin derivatives have electron microscope-detectable peroxidase activity. Hemin was coupled to several different size classes of dextran, to sucrose and to glucuronic acid. The peroxidase activity of the attached hemin was detected with the electron microscope using the method described by Graham and Karnovsky for horseradish peroxidase. These observations indicate that hemin may be used as a prosthetic group to label macromolecules for electron microscopic localization and raise the prospect of creating groups of inexpensive tracer substances of varying size, charge and specificity for histologic and ultrastructural studies.
HYDROXY-FERRIC IONS IN HISTOCHEMISTRY IRON ION HYDROXYLATION AND HISTOTOPOCHEMISTRY OF TISSUE IRON UPTAKE
Authors: LILLIE R. D. , VACCA LINDA , PIZZOLATO PHILIP
Published: April 2011

The form of iron which binds specifically to mucins in the Mayer (10) and Hale (2) reactions and their variants appears to be the complex ion FeOH++. The reagent is produced in two ways, the better by carefully computed H2O2 oxidation of ferrous chloride (or acetate in the Mayer reaction), the second by addition of NaOH to FeCl3 solutions, best at a 1:1 M ratio. The first reagent is relatively stable, an 0.1 M solution remaining useful for 4 months; 10 mM solutions of both are usable repeatedly for 4 and 3 weeks, respectively, for the 1st and 2nd variants. The binding of this form of iron is prevented by methylation, impaired variably by acetylation and unaffected by a deamination adequate to prevent anionic dye staining. These characters distinguish FeOH++ sharply from Fe++ and Fe+++ which bind to lysyl and arginyl residues and whose binding is prevented in lysyl areas by both methylation (amine alkylation) and deamination. The action on a number of specific mucosubstances in man and laboratory animals is described. Gastric epithelial mucoprotein is reactive (rat and guinea pig), in distinction from its nonreactivity to most metachromatic basic dyes (7). Reactivity was recorded for guinea pig tracheal cartilage and glands, duodenal goblet cells, Brunner gland mucin, crypt and goblet cell mucins of ileum and colon, for rat mast cells, sublingual and submaxillary but not parotid glands, for human umbilical cord matrix and nucleus pulposus.
THE ELASTIC FIBER A REVIEW
Authors: ROSS RUSSELL
Published: April 2011

A number of important questions remain to be answered concerning our understanding of elastic tissues. The size and molecular weight of the elastin precursor remains to be clearly established. The number of proteins involved in the microfibrillar component of the elastic fiber are as yet undetermined, although it would appear that they are glycoproteins that may represent a species of reasonably high molecular weight. Clearly the elastic fiber contains two morphologic components. During morphogenesis, the elastic fiber begins to appear in the form of aggregates of microfibrils that take the shape and direction of the presumptive elastic fiber. With increasing maturity elastin begins to form within the interstices of each bundle of microfibrils. By the time the elastic fiber is fully formed it consists largely of the amorphous component, elastin, surrounded by an envelope of microfibrils with microfibrils embedded within its interstices.

It has been suggested that the microfibrils form and take their shape extracellularly under the influence of the cells that have secreted their precursors. After the aggregates of microfibrils have taken their shape Ross and Bornstein (22) have suggested that the elastin may interact ionically with the surface of the microfibrils, since each of these two components has an opposite net charge, and may be held in position while desmosine cross-links are established through the action of the enzyme, lysyl oxidase. Thus the microfibrils would serve as a scaffolding to determine morphogenetically the shape and direction to be later taken by the mature elastic fiber.

The reason for the elastic properties of the elastin is still yet poorly understood, and the means by which the cells synthesize and secrete both of these components remain to be investigated.
DISTRIBUTION OF ANOMALOUS LYSOSOMES IN THE BEIGE MOUSE: A HOMOLOGUE OF CHEDIAK-HIGASHI SYNDROME
Authors: OLIVER CONSTANCE , ESSNER EDWARD
Published: April 2011

The beige mouse has been considered as a homologue of Chediak-Higashi syndrome in man. A light microscopic survey of 23 beige mouse tissues, using acid phosphatase as a lysosomal marker, revealed the presence of enlarged (anomalous) lysosomes, often in the form of aggregates, in 15 of the tissues examined. The degree of anomaly varied from one tissue to another and within cells of a given tissue. The most striking alterations occurred in liver parenchymal cells, kidney proximal tubule cells, Purkinje cells and granulocytes. Electron microscopy of liver and kidney revealed enlarged lysosomes containing numerous lipid-like inclusions. The widespread occurrence of anomalous lysosomes strengthens the homology between the beige mouse and Chediak-Higashi syndrome and further supports the concept that lysosomes are intimately involved in this disorder.
AN IMMUNOFLUORESCENT TECHNIQUE FOR OBSERVING THE BINDING OF CONCANAVALIN A TO FROZEN TISSUE SECTIONS
Authors: PRATT ROBERT M. , GIBSON WILLIAM A.
Published: April 2011

An indirect fluorescent antibody technique has been developed to observe the binding locus of concanavalin A (CON A) to specific structures in frozen tissue sections. The technique was developed using frontal sections of the embryonic rat head with particular attention focused on the secondary palate. The binding of CON A appeared to be mainly extracellular with the most intense fluorescence observed in basement lamina and cartilaginous structures. CON A binding was highly specific in that it was eliminated in the presence of competing sugars such as α-methyl mannoside. This relatively quick and simple technique should allow the observation of developmental or pathologic changes in CON A binding to frozen tissue sections from a variety of sources.
A METHOD FOR DETERMINING THE DISTRIBUTION OF LITHIUM IN WHOLE BRAIN SECTIONS BY USE OF 6Li(n,α)3H REACTIONS AND RADIOGRAPHIC DIELECTRIC TRACK REGISTRATION TECHNIQUES
Authors: NELSON S. C. , BARCHAS J. D. , BENSCH K. G. , HERMAN M. M.
Published: April 2011

A method for demonstrating the distribution of lithium in whole brain sections of the rat by using radiographic dielectric track registration techniques is described. The method essentially involves freeze-drying coronal sections, overlaying them with dielectric films sensitive to alpha particles, bombarding the tissue and film with thermal neutrons and chemically etching the lithium (6Li(n,α)3H) tracks in the films. The tracks in the film are superimposed on the tissue and the distribution of the lithium is delineated by the alpha particle tracks. The method is extremely sensitive and suitable for any tissue where lithium-induced tracks are significantly greater than the background and should be useful in elucidating some of the biologic properties of lithium.
DEVELOPMENT OF MUSCLE FIBERS IN THE COMPLEXUS MUSCLE OF NORMAL AND DYSTROPHIC CHICKS
Authors: ASHMORE C. R. , ADDIS P. B. , DOERR L. , STOKES H.
Published: April 2011

Musculus complexus in the chick hypertrophies prior to hatching, primarily because of lymph infiltration, then regresses in size after hatching. Growth resumes after 1 week of age. The population of β fibers is formed early in myogenesis and is completed prior to completion of a fiber formation. As in the fetal lamb, β fibers serve as structural centers around which a fibers develop. Cytochemical studies of chick muscle show that β fibers initially exhibit adenosine triphosphatase activity which is stable to both acid and alkaline treatment, but with further differentiation the activity is stable only to acid treatment. Only a small percentage of dystrophic chicks from 2 weeks to 3 years of age exhibits signs of muscle fiber degeneration in musculus complexus and then only a few fibers are affected. In contrast, in the pectoralis muscle of 3-year-old dystrophic chicks there are few, if any, fibers remaining which do not exhibit advanced stages of dystrophy. The medial adductor muscle, which has a fiber type composition comparable to that of musculus complexus, also exhibits myopathic changes which are severe, relative to those observed in musculus complexus of dystrophic chicks. Progression of hereditary muscular dystrophy in muscle fibers of the chick appears to be correlated with reduction of aerobic metabolic capacity and accompanying reduction of fiber activity.
THE HISTOCHEMICAL FLUORESCENCE METHOD FOR THE DEMONSTRATION OF CATECHOLAMINES THEORY, PRACTICE AND APPLICATION
Authors: FUXE KJELL , JONSSON GÖSTA
Published: April 2011

The formaldehyde fluorescence technique is a highly sensitive and specific method for the demonstration of catecholamines (CA) at the cellular level. It permits the demonstration of as little as about 5 x l0–4 pg noradrenaline, present in a single varicosity. The method can also be used for quantitative microfluorimetric studies on the CA stores at the cellular level, although a concentration-dependent quenching of the fluorescence in locations where the CA concentration is very high may impose some limitations. The sensitivity of the technique has been increased by using sections of unembedded tissue from unfixed or formalin-fixed tissue (Hökfelt and Ljungdahl). A glyoxylic acid technique, recently introduced by Axelsson and his co-workers, may prove to be an additional reliable alternative method for the demonstration of CA. Examples of neurobiologic problems that could be approached by using the formaldehyde fluorescence technique are briefly reviewed.
IMMUNOFLUORESCENCE OF DOPAMINE-β-HYDROXYLASE APPLICATION OF IMPROVED METHODOLOGY TO THE LOCALIZATION OF THE PERIPHERAL AND CENTRAL NORADRENERGIC NERVOUS SYSTEM
Authors: HARTMAN BOYD K.
Published: April 2011

This paper reviews key aspects of immunofluorescent methods for demonstration of dopamine-β-hydroxylase and discusses some recent applications of these methods.
A CHARACTERIZATION OF NUCLEOSIDE DIPHOSPHATASE IN THE ONION ROOT TIP
Authors: KLOHS W. D. , GOFF C. W.
Published: April 2011

Inosine diphosphatase of a Golgi-enriched fraction of the onion root tip was characterized. Peak enzyme activity occurred at pH 4.8 and 7.0, although considerable activity was present between the peaks. The activity at neutral pH approximately doubled during a 4-day cold storage; no such increase of the pH 4.8 activity occurred under similar conditions. Treatment with deoxycholate or Triton X-l00 also activated the pH 7.0 enzyme. Although magnesium, manganese and calcium all supported inosine diphosphatase activity, 2 mM manganese supported maximal activity. Uridine diphosphate, guanosine diphosphate and inosine diphosphate were hydrolyzed much more rapidly than the other substrates tested. Sodium fluoride, uranyl nitrate, potassium chloride and heat all partially inhibited the enzyme. Glutaraldehyde and lead nitrate, two reagents to which nucleoside diphosphatase is exposed when studied cytochemically, greatly reduced enzyme activity.
USE OF THE DIAZOSULFANILIC ACID, pH 1 AZURE A SEQUENCE ON ADRENAL MEDULLA AND THE EFFECTS OF PRIOR OXIDATION AND REDUCTION ON THE REACTION IN SEVERAL SPECIES
Authors: LILLIE R. D. , PIZZOLATO P. , VACCA L. L. , CATALANO R. A. , DONALDSON P. T.
Published: April 2011

With the diazosulfanilic acid, pH 1 azure A sequence adrenal medulla exhibits areas of pale green cells with green nuclei alternating with other areas grading from green to dark blue. Adrenal cortex and capsular collagen color greenish yellow, nuclei and elastin green, smooth muscle yellowish green and erythrocytes greenish brown to greenish black. The reaction is obtained best after 5% glutaraldehyde, then 10% formol then glyoxal, quite well with formol-free sublimate and lead acetate fixations and fails after Zenker and Kose bichromate formol fixations. Staining is prevented by prior oxidation (quinonization) with 10% iodine/methanol (1-2 hr 3°C), 0.1 M FeCl3 (2 hr), 3-5% K2Cr2O7 (2-3 hr) and 1% H5IO6 (10 min) and after these oxidations is restored by 4 hr 5% dithionite (N2S2O4), metabisulfite (Na2S2O5) or thiosulfate (Na2S2O3 · 5H2O) reductions. The reaction has been demonstrated in man (Homo sapiens), monkey (Macaca mulatta), hog (Sus scrofa dom.), dog (Canis familiaris), cat (Felis cattus), guinea pig (Cavia cobaya), rat (Rattus norvegicus albinus), mouse (Mus musculus albinus), gerbil (Meriones unguiculatus) and opossum (Didelphis virginiana). In the hog most of the adrenal medulla gave the dark blue reaction, fairly large areas with some segregation into islets of dark blue occurred in man, monkey, cat, dog and opossum and islets are seen in mouse, smaller in rat and inconstantly in the guinea pig and gerbil. It is thought that the reaction is showing noradrenaline. The method is not specific for this substance; certain proteins as hemoglobin and keratohyalin as well as enterochromaffin cells react strongly.
CHOLINESTERASE AND THE ATRIOVENTRICULAR NODE AND BUNDLE IN THE HUMAN FETUS UP TO MIDTERM
Authors: TAYLOR IAN M. , ANDERSON ROBERT H.
Published: April 2011

Both acetyl- and pseudocholinesterases have been detected in the atrioventricular specialized tissue of the human fetus. The upper part of the node, derived from sinus horn musculature, is cholinesterase-negative but contains acetylcholinesterase-positive nerves. The lower portion of the node and the common trunk of the bundle differentiate from atrioventricular canal muscle, are intrinsically pseudocholinesterase-positive and do not contain nerves up to the 190-mm crown-rump length stage. These observations are compared with those of previous workers who believed the specialized tissue of adult specimens of several species to contain only acetylcholinesterase. The possibility of a developmental change in the type of cholinesterase in the specialized tissue is discussed in relation to a recent study of the atrioventricular node of the neonatal rat.
THE QUANTITATIVE HISTOCHEMISTRY OF THE SYMPATHOADRENAL SYSTEM II. ENZYMES OF THE CITRIC ACID, PENTOSE PHOSPHATE AND GLYCOGENOLYTIC PATHWAYS
Authors: NELSON JAMES S.
Published: April 2011

The activities of enzymes from the citric acid cycle, pentose phosphate pathway and glycogen cycle were measured in collections of sympathoadrenal cells and compared with other neuronal groups. Enzymes from each pathway are found in groups of sympathetic neurons and adrenal medullary cells. The citric acid cycle has the highest capacity among glucose pathways in sympathoadrenal cells. Differences in glycolytic and citric acid cycle capacities between sympathetic ganglion and the other neuronal groups may be related to differences in capillary density. The capacity of the pentose phosphate pathway and reduced nicotinamide adenine dinucleotide phosphate-linked isocitric dehydrogenase is higher in sympathoadrenal cells than in the other cells surveyed. The enzyme activities in sympathetic ganglion are in excess of those needed to sustain the rates of glucose metabolism previously observed in this structure.
CHEMISTRY OF THE FILAMENTS AND TUBULES OF BRAIN
Authors: SHELANSKI MICHAEL L.
Published: April 2011

In this paper the main fibrous proteins of the nervous system are discussed from a biochemical standpoint. The biochemical properties of the proteins making up the neurofilaments and neurotubules are briefly reviewed and attention is turned to the assembly of supramolecular structures from tubulin, the microtubular protein. Vinblastine-induced assembly is surveyed as a model for assembly and the role of guanosine 5'-triphosphate in this is noted. The in vitro assembly conditions for microtubules recently introduced by Weisenberg are recounted and the role of calcium in controlling this is noted. The role of guanosine 5'-triphosphate and the roles it may and may not play are discussed in some detail as is the role of cyclic adenosine monophosphate. The evidence presented does not support a role for phosphorylation of the protein subunit in microtubule assembly A tentative model for the in vivo control of microtubule assembly and the possible relation of cyclic adenosine monophosphate and hormones such as insulin and nerve growth factor are presented.
DISSECTION OF WET TISSUE AND OF FREEZE-DRIED SECTIONS IN THE INVESTIGATION OF SEMINIFEROUS TUBULES AND INTERSTITIAL TISSUE FROM RAT TESTIS
Authors: ROMMERTS F. F. G. , COOKE B. A. , VAN DER MOLEN H. J. , VAN DOORN L. G. , GALJAARD H.
Published: April 2011

Seminiferous tubules and interstitial tissue were dissected out from freeze-dried sections and from wet tissues of the rat testis. The results of these preparation procedures were compared in regard to the distribution of a nonspecific esterase activity and of radioactive labeled steroids. Nonspecific esterase activity was found 50 times higher in interstitial tissue than in the seminiferous tubules, when samples disssected from wet tissue were analyzed. When specimens from freeze-dried sections were used for assays, this ratio was somewhat lower. In normal rat testes, the amount of interstitial tissue varied from 13 to 23%. The percentage of interstitium increased to about 50% in rats fed a diet lacking essential fatty acids. In seminiferous tubules isolated by the wet dissection technique there was no indication of the presence of interstitial tissue. Both fractionation procedures are useful in the analysis of enzyme activities, but the dry dissection method is preferable for studying the distribution of diffusible compounds, like steroids, because during wet dissection some redistribution of labeled steroids did occur.
ANALYSIS OF SUBMICROSCOPIC STRUCTURES BY THEIR EMITTED X-RAYS
Authors: DEMPSEY E. W. , AGATE F. J. , LEE M. , PURKERSON M. L.
Published: April 2011

X-ray emission spectra have been recorded from several biologic tissues using a multichannel energy-dispersive analyzer with a retractible semiconductor detector coupled to a Cambridge Mark II scanning electron microscope. Particular attention has been given to the detection of silver in experimental argyria, of calcium in dermoid scales and in experimental necrosis of the kidney and of sulfur in the inner and outer portions of reptilian skin. Sulfur and chlorine have been found associated with silver in argyria. Phosphorus was associated with calcium both in the dermal scales and in necrotic areas. In addition to these elements, trace amounts of copper, zinc, lead, sodium, potassium, iron, arsenic, osmium and uranium have been detected in various normal and experimental situations. The applicability of the combined instrument to cytochemical problems is briefly discussed.
A NEW METHOD FOR THE INVESTIGATION OF THE KINETICS OF THE CAPTURE REACTION IN PHOSPHATASE CYTOCHEMISTRY I. THEORETICAL ASPECTS OF THE LOCAL FORMATION OF CRYSTALLINE PRECIPITATES
Authors: CORNELISSE C. J. , VAN DUIJN P.
Published: April 2011

Factors governing the precipitation kinetics and localization of lead phosphate in phosphatase cytochemistry are discussed, with special attention to the origin of diffusion artifacts. The principal differences between the kinetics of coupling reactions leading to insoluble organic dyes and of lead salt methods are treated in terms of the physicochemical theory of crystallization of sparingly soluble salts. This process, since it involves nucleation and crystal growth, is dependent on supersaturation and the presence of heterogeneous catalysts in the reaction medium. Under cytochemical conditions in tissue sections these factors are partially determined by the lead concentration in the medium and the local specific enzyme activity. It is postulated that heterogeneous nucleation of phosphate ions which have escaped from their enzymatic production sites may be a source of falsely localized precipitates. The need for experimental models for the study of the dynamics of precipitation and diffusion is stressed.
A QUALITATIVE AND QUANTITATIVE HISTOCHEMICAL INVESTIGATION OF THE EFFECTS OF PILOCARPINE STIMULATION ON AMYLASE LEVELS IN RAT SUBMAXILLARY AND PAROTID GLANDS
Authors: SHEAR MERVYN , GIBSON SHEILA , VAN DER MERWE ELIZABETH
Published: April 2011

A histochemical study, using the starch substrate film technique, was done to compare amylase activity in fresh-frozen cryostat sections of paired male rat parotid and submaxillary-sublingual glands, before and after pilocarpine stimulation. A quantitative procedure was followed on the same material. Amylase depletion was apparent in sections of parotid gland incubated on starch films for short periods. Quantitative estimations on adjacent sections showed an amylase depletion of about 50%. In the submaxillary-sublingual gland, amylase was demonstrable in the blood vessels in both unstimulated and stimulated material and there appeared to be a higher concentration of the enzyme in the latter. Quantitative assays on adjacent sections demonstrated a very considerably raised level of amylase activity in the stimulated submaxillary gland. It was concluded that the histochemically demonstrable amylase in the blood vessels of the submaxillary and sublingual glands was blood amylase and that the raised level following pilocarpine stimulation might be interpreted as a passage to the blood of enzymes produced by other glands under the same stimulus.
QUANTITATIVE ASPECTS OF CYTOCHEMICAL PEROXIDASE PROCEDURES INVESTIGATED IN A MODEL SYSTEM
Authors: STREEFKERK J. G. , VAN DER PLOEG M.
Published: April 2011

In many immunohistochemical studies horseradish peroxidase (HRP) is used as the marker enzyme. An in situ and indirect quantitation of antigen or antibody would be possible by determination of the HRP activity. For this reason quantitative aspects of histochemical reaction procedures to localize HRP activity have been studied in a model system consisting of polyacrylamide films into which the enzyme had been incorporated. A linear relation between the amount of HRP activity and the spectrophotometrically determined amount of reaction product could be established for reactions using 3,3'-amino-9-ethylcarbazole as the hydrogen donor. A similar relationship could not be found after staining with benzidine-containing media. These results indicate that for certain substrates the quantities of dyes formed during the cytochemical HRP reaction can be considered as a measure for the activity of the enzyme. This opens perspectives for the in situ quantitation of antigens or antibodies.
HUMAN LEUKEMIC CELLS CYTOCHEMICAL STUDIES ON ACIDIC NUCLEAR PROTEINS
Authors: DESAI L. S. , FOLEY G. E.
Published: April 2011

Utilizing staining with alkaline fast green (pH 8.1) and toluidine blue O (pH 9.0) stains, the nuclear basic and acidic protein content of individual human lymphocytic cells has been quantitated cytochemically with high resolution, rapid scanning instruments. There were distinct differences in the distribution patterns of the acidic and basic proteins in these human lymphocytic cells. Nucleoli were densely stained by toluidine blue O, confirming the presence of acidic proteins. The acidic nuclear proteins:DNA and basic nuclear proteins:DNA ratios are consistent and quantitatively similar, irrespective of the diagnostic category from which the cells derived and there was a definite correlation between nuclear proteins and DNA content. However, the acidic nuclear proteins represented a larger proportion of the total dry mass of the cell than did the basic nuclear proteins, an observation which may prove to be of interest with respect to control mechanisms.
ROBUST METAL COMPLEXES, FERROCENYLMETHYL CARBOXYHYDRAZIDE AND 1-CHLOROMERCURIFERROCENE, AS ELECTRON-OPAQUE STAINS FOR ALDEHYDES AND THIOL GROUPS
Authors: ALLEN DAVID E. , PERRIN DOUGLAS D.
Published: April 2011

Ferrocenylmethyl carboxyhydrazide has been used for the ultrastructural localization of glycogen, the glycocalyx of cat intestine and deoxyribonucleic acid. l-Chloromercuriferrocene is introduced as a stain for thiol groups in wool. Both compounds are examples of a potentially useful class of cytochemical stains made from a "robust" metal complex by inserting a suitable functional group on the periphery of the molecule. In such stains, where the metal is firmly bound, the need to carry out chelation reactions in the tissues is avoided and the risk of random metal deposition is greatly diminished.
THE ANATOMICAL CHARACTERISTICS OF DOPAMINE-β-HYDROXYLASE ACCUMULATION IN LIGATED SCIATIC NERVE
Authors: HARTMAN BOYD K.
Published: April 2011

The anatomical characteristics of the accumulation of dopamine-β-hydroxylase (DBH) that occurs after ligation of rat sciatic nerve were examined in detail using a recently developed specific immunofluorescence method. Although rapid accumulation of DBH occurred showing the presence of axonal transport, the anatomical observations indicated that the build-up was considerably more complex than the simple "blockade" model frequently used as a basis for computation of transport rates from quantitative estimates of accumulation. During the early time period (7 hr) the situation was complicated by a substantial amount of accumulation of DBH distal to the ligation. By 24 hr after ligation the distal accumulation was small compared to that present proximally. The size of the discrete areas covered by DBH fluorescence at 24 hr indicated that axonal membranes may have already started to rupture, thus releasing vesicles into the extracellular space. By 48 hr after ligation the DBH was no longer confined to the area near the ligation, but extended 3.6 mm proximally. By 96 hr after ligation there had been a net loss of DBH fluorescence indicating that degradation and clearance of accumulated DBH had occurred.