bluuebunny/crossref_metadata_2025 huggingface dataset:
A HISTOCHEMICAL EVALUATION OF THE PYROANTIMONATE-OSMIUM REACTION
Authors: CLARK MICHAEL A. , ACKERMAN G. ADOLPH
Published: April 2011

The chemical nature of the pyroantimonate-osmium (PAO) reaction in normal human bone marrow cells has been evaluated by the application of a number of digestion and blocking procedures as well as the electron microprobe. Five foci of reactivity are localized in cells fixed directly in the PAO reagent, viz., nucleoli, heterochromatin, cytoplasmic granules, particulate glycogen and the outer plasmalemmal surface. Heterochromatin and nucleolar staining are attributable to calcium bound to nucleic acids as well as to reactive amino groups on histones. Granule staining is dissociable into acid-dialyzable, ribonuclease (RNase)-stable and RNase-labile components but sulfate groups of acid mucosubstances are probably not involved. Glycogen staining by the PAO reagent has been verified and shown to result from the formation of organometallic complexes between the pyroantimonate ion and the C2-C3-free hydroxy groups of the glucose residues. PAO reactivity has been definitely localized along the outer plasmalemmal surface, but this component of PAO staining is resistant to all control measures employed. These studies have illustrated the complexity and polyvalency of the PAO reaction and have shown that PAO staining is not exclusively associated with metallic cation localization.
SOLUBLE ACIDIC LIPOPROTEINS OF BOVINE NEUROSECRETORY GRANULES RELATION TO NEUROPHYSINS
Authors: MYLROIE ROBERT , KOENIG HAROLD
Published: April 2011

Purified neurosecretory granules (NG) from bovine neurohypophysis stain metachromatically with acridine orange. NG were sonicated in 0.2% Triton X-100. The soluble protein, 83% of the total protein, was separated by ultracentrifugal flotation in KBr into two lipoprotein (LP) fractions, a high density lipoprotein fraction (HDLP, D < 1.35 g/ml), 37%, and a very high density LP fraction (VHDLP, D > 1.35 g/ml), 63% of the total. The HDLP contained 0.46 mg phospholipid and 0.13 mg cholesterol/mg protein. The VHDLP contained 0.08 mg phospholipid and 0.025 mg cholesterol/mg protein. The amino acid composition of the delipidated LPs resembled that of bovine neurophysins. Disc gel electrophoresis resolved two major and four minor components of identical mobilities in HDLP and VHDLP. The major anodic components, 90% of the protein, and one minor cathodic component were sudanophilic, reduced iodine and stained metachromatically with acridine orange. The NG lysates prepared without detergent or sonication contained 52% of the total protein and phospholipid. The insoluble residue consisted of membrane profiles and had a different lipid and amino acid composition. Gel electrophoretograms of water extracts of NG revealed the same protein components present in Triton X-100 extracts. The major anodic bands and the cathodic band were colored by Sudan black B and iodine and stained metachromatically with acridine orange. Prior extraction of gels with chloroform-methanol (2/1, v/v) abolished these staining reactions. Neurophysins prepared by extraction of acetone-dried tissue with 0.1 N HCl contained about 7% of the phospholipid present in Triton X-100 extracts. Gel electrophoretograms of neurophysins disclosed the same protein constituents seen in Triton X-100 and water extracts of NG. The major components did not stain with Sudan black B or iodine but stained metachromatically with acridine orange due to the presence of residual phospholipid. Neurophysins are essentially apoproteins of these soluble acidic LPs.
SEMIQUANTITATIVE ESTIMATION OF FORMALDEHYDE-INDUCED FLUORESCENCE OF NORADRENALINE IN CENTRAL NORADRENALINE NERVE TERMINALS
Authors: LIDBRINK PETER , JONSSON GÖSTA
Published: April 2011

The fluorescence-concentration relationship of noradrenaline (NA) in NA nerve terminals of rat cerebral cortex and hypothalamus demonstrated with the Falck-Hillarp formaldehyde fluoresence method has been investigated. Comparisons were made between experimentally induced changes in NA concentrations as recorded by chemical-analytical assay and fluorescence intensities of NA nerve terminals as estimated subjectively in the fluorescence microscope. The results obtained show that in cerebral cortex there was a close correlation between NA concentration and fluorescence intensity up to the normal endogenous NA level, indicating a linear relationship between the two parameters. In hypothalamus, however, a correlation between fluorescence intensity and NA concentration was observed only when the nerves contained less than 50% of the endogenous NA level. Above this value, there was no further proportional increase in fluorescence intensity, probably because of a concentration-dependent quenching of the fluorescence. Essentially the same results were obtained when investigating previously emptied NA nerve terminals refilled with various amounts of 3H-NA. The data presented clearly show that a good NA quantitation of cortical nerve terminals can be made by careful subjective estimation of the fluorescence intensity.
AN ULTRACYTOCHEMICAL AND ULTRASTRUCTURAL STUDY OF EPITHELIAL CELLS IN DUCTULI EFFERENTES OF CHINESE HAMSTER
Authors: YOKOYAMA MASAO , CHANG JEFFREY P.
Published: April 2011

Ultrastructural and ultracytochemical features of the efferent duct of Chinese hamster have been studied. The ducts are composed of two main types of epithelial cells, ciliated and nonciliated. Distinct structural and cytochemical characteristics of these cells are apparent. Presence of fibrogranular complex which is supposedly related to basal body replication was demonstrated in ciliated cells for the first time in this tissue. Thiamine pyrophosphatase activity in Golgi apparatus, acid phosphatase activity in Golgi apparatus and lysosomes and alkaline phosphatase activity on basal plasma membranes of both ciliated and nonciliated cells have been localized. However, thiamine pyrophosphatase activity was seen only on the luminal surface, apical vacuole and apical tubular structure of nonciliated cells but not on the surface of ciliated cells. Similarly, horseradish peroxidase was absorbed only by nonciliated cells. The cytochemical and ultrastructural differences between the two types of cells indicate a functional specialization. The results indicate that the ciliated cells are concerned with the transportation of the sperms and that the nonciliated cells are concerned with the regulation of fluid composition in the duct since the latter are capable of both secretion and absorption.
ELECTRON MICROSCOPIC LOCALIZATION OF GLYCOPROTEINS IN PITUITARY CELLS OF DUCK AND QUAIL
Authors: TIXIER-VIDAL ANDRÉE , PICART RENÉE
Published: April 2011

Structures demonstrating the presence of glycoproteins, acid phosphatase activity and OsO4 impregnation were localized by means of the electron microscope in duck and in quail pituitary cells. Two methods for the electron microscopic demonstration of glycoproteins were used: a chromic acid-phosphotungstic acid mixture on glycol-methacrylate-embedded tissues, and the periodic acid-thiocarbohydrazide-silver proteinate technique. Both methods showed glycoproteins in the following sites: ( a) the secretory granules in three types of cells (A, B, C) which are part of the seven different cells of the avian pituitary; ( b) the several kinds of dense bodies which are richer in reaction product than the secretory granules. A correlation with previous studies on similar species of birds is helpful in identifying each of the three positive types of cells as thyrotropic cell (A), prolactin cell (B) and gonadotropic cell (C). The presence of glycoproteins within the Golgi saccules (on condensing granules) was found with the periodic acid-thiocarbohydrazide-silver proteinate method in these gonadotropic cells only. In gonadotropic and thyrotropic cells, acid phosphatase activity is weak in the inner Golgi saccules and strong in the "Golgi Endoplasmic Reticulum Lysosomes" system, in the lysosomes, in the dense bodies and in the vacuolated dense bodies. The structures which are richest in glycoproteins are also those which have the most acid phosphatase activity. On the contrary, OsO4-stained structures in duck gonadotropic cells (nuclear pericisterna, rough endoplasmic reticulum, cisternae and outer Golgi saccules) have no glycoproteins or acid phosphatase activity.
NONSPECIFIC ESTERASES IN THE MYONEURAL JUNCTION OF HUMAN STRIATED MUSCLE
Authors: CHOKROVERTY S. , PARAMESWAR K. S. , CO C.
Published: April 2011

By the use of acetyl- and butyrylthiocholine and α-naphthyl acetate as substrates and selective inhibitors, we have shown the presence of both specific and nonspecific esterases in the myoneural junction of vastus medialis and peroneus brevis muscles of normal subjects as well as in paretic muscles of hemiplegic patients. The presence of nonspecific esterases in human myoneural junction has not been recorded previously. The nonspecific esterases in our fixed tissue were noted to be predominantly E600-sensitive at 10–5 M concentration although resistant at 10–6 M and 5 x 10–6 M concentration.
ULTRASTRUCTURAL LOCALIZATION OF SUCCINATE DEHYDROGENASE IN MOUSE LIVER MITOCHONDRIA; A CYTOCHEMICAL STUDY
Authors: KALINA MOSHE , WEAVERS BARRY , PEARSE A. G. E.
Published: April 2011

Ultrastructural localization of succinate dehydrogenase, employed as a marker of the electron transfer chain, was studied in mouse liver mitochondria either in orthodox or condensed conformation. Activity of succinate dehydrogenase in the orthodox form mitochondria was localized primarily on the cristal membrane facing the matrix. A prolonged incubation time resulted in reaction product filling the intracristal space, whereas a short incubation time resulted also in a product which appeared mainly at opposite points on both sides of the crista. No such arrangement of the reaction product was noted in mitochondria in the condensed conformation. These findings, supported by studies of serial sections, suggest that the reaction product in orthodox form mitochondria appears in bands or coils surrounding the whole cristal membrane.
CYTOCHEMICAL LOCALIZATION OF MYOFIBRILLAR ADENOSINE TRIPHOSPHATASE ACTIVITY IN SARCOMERES OF GLYCERINATED MUSCLE BY THE CALCIUM PRECIPITATION METHOD A NOVEL CONTROL IS NEEDED TO DEMONSTRATE ARTIFACTS
Authors: TUNIK BERNARD D.
Published: April 2011

The calcium precipitation method was used in an attempt to localize myofibrillar adenosine triphosphatase (ATPase) activity within the sarcomeres of single, unfixed, glycerol-extracted fibers from the pectoralis major muscle of leghorn chicks. ATPase was apparently present in only the A-I overlap region of unshortened fibers, and was apparently present in only the contraction bands of shortened fibers. The results are shown to be self-contradictory in view of the known submicroscopic morphology of the sarcomeres. However, the usual classical controls provided no evidence of artifactual localization. In a novel control, separate solutions of calcium and phosphate were caused to diffuse simultaneously into a fiber. The resulting precipitate was localized in patterns indistinguishable from those obtained from incubation of shortened or unshortened fibers in ATP. These patterns result from the localization of precipitate in regions of high protein concentration. Caution in interpreting the results of cytochemical procedures even though controls provide no evidence of artifactual localization is urged.
ELECTRON STAINING WITH URANYL ACETATE POSSIBLE ROLE OF FREE AMINO GROUPS
Authors: LOMBARDI LUCIANO , PRENNA GIOVANNI , OKOLICSANYI LAJOS , GAUTIER ALAIN
Published: April 2011

Uranyl acetate (UA) is known to have affinity for nucleic acids and –COOH groups. Experiments were performed to test the possible affinity of UA for free amino groups. For this purpose, –NH2 blocking and deamination techniques (with dinitrofluorobenzene, nitrous acid and Chloramine T) were applied to form-aldehyde-fixed pancreas samples, after which the stainability of zymogen granules by UA was strongly reduced. It is concluded that free amino groups may also participate in the binding of UA in biologic specimens.
DISCREPANCIES BETWEEN CYTOPHOTOMETRIC FEULGEN VALUES AND DEOXYRIBONUCLEIC ACID CONTENT
Authors: NOESKE K.
Published: April 2011

Diploid nuclei of different cell types of the granulocytopoietic and erythropoietic series showed different Feulgen values. The highest values were found in the most immature nuclei, and the lowest values in the most mature cells. Extraction of acid-soluble nuclear proteins brought the different values to the same level. Examples of similar findings in other cell types, as described in the literature, are discussed. The Feulgen reaction and its colored product depend on the functional state of the chromatin in such a way that nuclei with high template activity of deoxyribonucleic acid show high Feulgen values whereas mature, dense nuclei with low template activity have lower Feulgen values, although both kinds of nuclei contain identical amounts of deoxyribonucleic acid.
ACID CATALYSIS OF THE FORMALDEHYDE CONDENSATION REACTION FOR A SENSITIVE HISTOCHEMICAL DEMONSTRATION OF TRYPTAMINES AND 3-METHOXYLATED PHENYLETHYLAMINES 2. CHARACTERIZATION OF AMINE FLUOROPHORES AND APPLICATION TO TISSUES
Authors: BJÖRKLUND ANDERS , NOBIN ANDERS , STENEVI ULF
Published: April 2011

A histochemical fluorescence method is presented in which a controlled activation of the condensation reaction between formaldehyde and certain aromatic monoamines is achieved by the introduction of minute amounts of HCl gas in the formaldehyde reaction vessel. In this way, not only highly reactive catecholamines and 5-hydroxytryptamine but also less reactive compounds, such as tryptamine, 5-methoxytryptamine and 3-methoxylated phenylethylamines which give only weak fluorescence in the noncatalyzed formaldehyde reaction, can be converted into intensely fluorescent products. The acid-catalyzed formaldehyde treatment proved to be a highly sensitive technique for the demonstration of these various monoamines in freeze-dried tissues. The reaction thus proceeded without any observable dislocation of the substances from their cellular stores and the resulting fluorophores were well visible against an essentially dark background. The procedure was successfully, and equally well, applied to whole freeze-dried tissue pieces, to paraffin sections of freeze-dried specimens and to freeze-dried cryostat sections. The fluorescence properties of a large number of phenylethylamine and indolylethylamine fluorophores, formed in the acid-catalyzed formaldehyde reaction, have been investigated microspectrofluorometrically in models and in freeze-dried tissues. The fluorescence spectra of the amine fluorophores were consistent and fully reproducible, making possible the accurate characterization of cellular monoamines with this method.
FINE STRUCTURAL IDENTIFICATION OF PEROXISOMES IN MOUSE AND RAT BRONCHIOLAR AND ALVEOLAR EPITHELIUM
Authors: PETRIK PETR
Published: April 2011

Following incubation in Novikoff and Goldfischer's alkaline modification of Graham and Karnovsky's diaminobenzidine (DAB) medium, densely stained bodies were identified in the bronchiolar nonciliated cells (Clara cells) and in type II alveolar cells (great alveolar cells, granular pneumonocytes) in mouse and rat lung. In Clara cells, the electron-dense reaction product was distributed in spherical, single membrane-bound bodies having an average diameter of 0.3-0.4 µ but varying from 0.15-0.65 µ. Neither nucleoids nor crystalline cores were seen inside these structures. These bodies were seen to be often closely associated with rough endoplasmic reticulum cisternae. The electron-lucent vesicles, some of them with a dense core, previously reported as secretion granules, never stained with DAB. The DAB-stained bodies present in type II alveolar cells had a somewhat different morphology. They were often elongated, with an average size of 0.2-0.3 x 0.1 µ but varying between 0.6 and 0.08 µ. The electron density of their staining was the same as in the DAB-positive bodies of Clara cells. Both cell types were studied for acid phosphatase activity; the distribution pattern of the reaction was entirely different from the one reported above. Considering the specificity of this modified reaction and the effect of inhibitors, it is assumed that the DAB-stained bodies present in both Clara and type II alveolar cells—in spite of a slightly atypical aspect in the latter—are peroxisomes.
CYTOCHEMICAL LOCALIZATION OF CHOLINESTERASE ACTIVITY IN ADULT RABBIT HEART
Authors: HAGOPIAN MARTIN , TENNYSON VIRGINIA M.
Published: April 2011

The papillary muscle of the adult rabbit heart was studied by a modification of the Koelle-Friedenwald copper thiocholine technique for the localization of cholinesterase activity. Butyrylcholinesterase (BuChE), identified by its substrate and inhibitor specificity, is found mainly in the terminal sacs of the sarcoplasmic reticulum adjacent to the T system. The localization of the reaction product in this particular site suggests that BuChE may play a role in excitation-contraction coupling in the adult rabbit heart. The present findings are also discussed in comparison with our previous work on the localization of acetylcholinesterase activity in the embryonic rabbit heart.
GLUCOSE 6-PHOSPHATASE ACTIVITY IN THE INTESTINAL EPITHELIUM OF THE MOUSE
Authors: HUGON J. S. , MAESTRACCI D. , MÉNARD D.
Published: April 2011

Glucose 6-phosphatase activity has been studied in the mouse intestinal tract using biochemical, cytochemical and cytophotometric methods. The enzyme is present in all segments of the digestive tract including the colon and is essentially localized in the absorbing cells. The maximum of activity is recorded in the middle jejunum. In each intestinal segment, the median part of the villi exhibits the highest reaction. In the epithelial cells, the enzyme is observed in the rough and smooth endoplasmic reticulum and in the nuclear envelope. Cisternae of the Golgi apparatus are always negative. This enzyme appears to be a useful tool to study the regulating mechanisms operating in the differentiating epithelial cells of the intestine after an appropriate stimulation.
THE PRESENCE OF LIPID DROPLETS AND THE ABSENCE OF STABLE SUDANOPHILIA IN OSMIUM-FIXED HUMAN LEUKOCYTES
Authors: COIMBRA ANTONIO , LOPES-VAZ A.
Published: April 2011

In blood smears fixed in osmium vapor and stained with Sudan black B or oil red O, neutrophil and eosinophil leukocytes contain a regular number of sudanophilic granules and do not exhibit stable sudanophilia. Stable sudanophilia occurs following aldehyde fixation without the appearance of sudanophilic granules in white cells. The sudanophilia in granules was lysochromic since the granules could be reversibly decolorized by dye solvents. Cold acetone extracted the content of the granules following osmium tetroxide oxidation. Electron microscopically the granules had the appearance of typical lipid droplets. Human granulocytes thus contain a constant amount of free lipid deposits that are adequately preserved only by osmium tetroxide fixation. Osmium seems to prevent the occurrence of stable sudanophilia through inhibition of myeloperoxidase activity. The procedure may be useful to follow variations of lipid content in human leukocytes, as exemplified by the excess of lipid found in leukocytes, in synovial fluids of patients with rheumatoid arthritis.
EFFECTS OF INFLAMMATION ON SUBPOPULATIONS OF BLOOD LYMPHOCYTES
Authors: WIENER JOSEPH , GIACOMELLI FILIBERTO , POMERANZ JOANNA V. , LOUD ALDEN V.
Published: April 2011

A method for quantitative cytochemistry has been used to study the surface properties of blood lymphocytes of guinea pigs 24 hr after the production of cell-mediated immunity or acute inflammation. Three subpopulations of small lymphocytes with different surface-binding characteristics for Thorotrast particles have been observed. The average number of particles per micron length of surface membrane, i.e., particle density, has been determined for each class of cells. Only two clusters separated by a distinct gap are apparent in each group of animals. There is a significant decrease in the mean particle density of the major subpopulation of lymphocytes in each group of animals with inflammatory reactions, thereby suggesting a reduction in surface charge. On the other hand, the particle density distribution of the minor population of cells does not differ from normal. The role of surface charge alterations on the circulation, margination and emigration of lymphocytes is discussed.
CONTROLLED CHROMATION
Authors: ELFTMAN HERBERT
Published: April 2011

Controlled chromation represents another step in the development of dichromate oxidation as a cytochemical technique, to which previous contributions have been made by Weigert, Smith, Dietrich, Ciaccio and Baker. Recognition of the critical importance of controlling pH, in addition to the regulation of temperature, time, and concentration, allows voluntary adjustment of these variables to fit each particular problem. Control of chromation is equally important whether it is exercised on fresh tissues, frozen sections, or tissues previously subjected to the action of fixatives or solvents.

By using graded intensities of chromation, differential visualization of a series of cell constituents may be achieved. Judicious combination of controlled chromation with the use of other reagents, including solvents, offers the possibility of developing analytical methods for the study of a great variety of lipid and non-lipid tissues components.

The relative ease with which phospholipids, such as those of the Golgi apparatus, are chromated allows their routine visualization to be accomplished after chromation at pH 3.5 for 18 hours at 56°C. with 2.5% potassium dichromate, followed by paraffin embedding. Alternate sections stained with Sudan black B and with hematoxylin can be ready for study within twenty four hours of the collection of the tissue. Chromated lipids are stained by both Sudan black B and by hematoxylin, non-chromated lipids only by Sudan black B, and non-lipid chromated substances only by hematoxylin. Other dyes may be used in place of Sudan black B and hematoxylin when advantageous.

The staining of chromated tissue with hematoxylin can be controlled by the addition of any one of several reagents to the staining solution, so as to restrain the formation of hematoxylin lakes by minor concentrations of chromium. Differentiation is consequently accomplished during staining and the entire process can be completed on paraffin sections at 56°C. in two hours.
THE NATURE OF THE REACTION BETWEEN ORCEIN AND ELASTIN
Authors: WEISS JULES
Published: April 2011

Available data are consistent with the interpretation that the reaction between orcein and elastin is due to the formation of hydrogen bonds. The selectivity for elastin of this reaction in acid alcohol solution is probably due to the uniquely low positive charge of elastin in such solutions. The use of alcohol solutions is necessary to solubilize the positively charged orcein fractions.
HISTOCHEMICAL DEMONSTRATION OF PROTEIN-BOUND AMINO GROUPS
Authors: WEISS LEON P. , TSOU KWAN-CHUNG , SELIGMAN ARNOLD M.
Published: April 2011

A method for the histochemical demonstration of protein-bound amino groups is described. The method is based upon the reaction of primary amino groups with aldehydes to form Schiff bases. By the use of 3-hydroxy-2-naphthaldehyde, a red to blue azo dye is formed at the sites of reactivity in fixed tissue sections, after coupling with tetrazotized diorthoanisidine. Preparations of the reagent, histochemical procedure, evidence for the specificity of the reaction and the results of its application to normal rat tissues are given.
ATTEMPTS TO DEMONSTRATE ACETYLCHOLINESTERASE ACTIVITY IN BLOOD AND BONE-MARROW CELLS BY A MODIFIED THIOCHOLINE TECHNIQUE
Authors: ZAJICEK J. , SYLVEN B. , DATTA N.
Published: April 2011

The thiocholine method of Koelle (1951) was used in attempts to demonstrate acetylcholinesterase activity in artificial enzyme-gelatin mixtures and in suspensions and smears of isolated platelet, ghosts, erythrocytes, and megakaryocytes of different species. When the last processing step with ammonium sulphide was omitted, the following observations were made:

After a short time of incubation, small granular or rod-like deposits were found at the presumed sites of AChE activity. These deposits apparently grew into large crystals during prolonged incubation. Such crystals were demonstrated at the surface of platelet ghosts and erythrocytes of species bearing AChE activity, and further in megakaryocytes of some species presumably bearing this activity. The crystals were provisionally assumed to be a Cu thiocholine salt representing sites of AChE activity. If an enzyme inhibitor was added or a substrate used which is not split by AChE no crystals appeared.
THE EFFECT OF COMA ON THE ENZYMES OF THE BRAIN: HISTOCHEMICAL AND BIOCHEMICAL STUDY
Authors: CHESSICK RICHARD D.
Published: April 2011

Histochemical and quantitative chemical evidence is presented that coma induced by pentobarbital, insulin, or anoxia does not affect the distribution of concentration of acid phosphatase, alkaline phosphatase, phosphamidase, 5-nucleotidase, α-naphthol esterase and naphthol AS esterase in the brain of the rat. Soulairac's findings that the alkaline phosphatase activity is markedly decreased in coma have not been confirmed. The interpretation of Swank and Cammermeyer's reported shift in the acid phosphatase concentration is open to doubt.
THE HISTOCHEMICAL DISTRIBUTION OF 5-NUCLEOTIDASE AND UNSPECIFIC ALKALINE PHOSPHATASE IN THE TESTICLE OF VARIOUS SPECIES AND IN TWO HUMAN SEMINOMAS
Authors: WACHSTEIN M. , MEISEL E.
Published: April 2011

Sections of human adult testicles and of testicles from dog, rabbit, guinea pig, rat and mouse as well as of two seminomas were stained for nonspecific alkaline phosphatase and for 5-nucleotidase. The most outstanding feature in the testicles of all species examined is the presence of 5-nucleotidase predominantly located in the nuclei of the spermatogenic elements. In the two seminomas 5-nucleotidase was likewise found in the nuclei of the tumor cells. The validity of these findings is discussed and it is concluded that the predominantly nuclear localization reflects the true distribution of 5-nucleotidase in these cells.

The seminomas are not only rich in 5-nucleotidase, but contain apparently considerably larger amounts of nonspecific alkaline phosphatase than do normal human spermatogenic cells. The seminomas, therefore, show a distinct staining pattern as regards 5-nucleotidase and alkaline phosphatase so far apparently not observed in other tumors.
MICRO X-RAY DIFFRACTION TECHNIQUES FOR USE IN HISTOCHEMISTRY
Authors: CARLSTRÖM D.
Published: April 2011

General considerations are given of the use of microdiffraction techniques in biological work. A cylindrical microcamera which permits registration of rotation photographs, as well as flat plate and fibre photographs is described. It has been successfully applied in the study of the crystallographic structure of single statoconia from man and some vertebrates. Rotation patterns from parts of single haversian systems in bone tissue and from thin splits of tooth enamel have also been recorded.
THE EFFECT OF METHYLATION ON BASOPHILIA
Authors: FISHER EDWIN R. , LILLIE R. D.
Published: April 2011

A mixture of 0.05 N HCl and methanol is capable of completely inhibiting thionin and azure A metachromasia of intestinal mucin, cartilage and mast cell granules after treatment for 2 hours at 58°C. or 48 hours at room temperature. Slight reduction is noted after 2 weeks at 3°C.

Cytoplasmic basophilia is completely destroyed after 4 hours of methylation at 58°C. or 48 hours at room temperature. Pancreatic acinar cell cytoplasm appears more resistant, requiring 8 hours at 58°C. and longer than 9 days at room temperature. Methylation at 3°C. is ineffective in inhibiting this basophilia even after 2 weeks.

Nuclear and nucleolar basophilia with aniline dyes (azure A and thionin) requires 24 hours of methylation at 58°C. or 14 days at room temperature before there is complete destruction. There is no effect produced by methylation at 3°C. for 2 weeks. Alum hematoxylin staining of these structures is unaffected by the methylation procedure, indicating the different nature of action of this dye complex from the aniline dyes used.

Reversal of the methylation procedure or demethylation can be accomplished with 0.5% KMnO4 for ½ hour at room temperature provided the methylation has not been too prolonged. Other oxidants such as periodic, peracetic and chromic acids as well as aqueous HCl and alkali (borax solution) fail to reverse the blockade.

The substitution of 0.1 N HCl for the usual 0.05 N HCl in the methylation procedure brings about effective blockade in approximately ½ the time required by the 0.05 H NCl methanol.

Despite the removal of cytoplasmic and nuclear basophilia by this method sections treated concomitantly and stained with the Feulgen nucleal procedure demonstrate strong Feulgen reactions indicating selectivity for the acid portion of the nucleic acid involved.

Methylation after fixation of tissue in a chromate containing fixative (Zenker acetic) requires more prolonged treatment to inhibit cytoplasmic basophilia than is required after Formalin or 95% alcohol fixation. Otherwise the reactions occur similarly after either of the three fixatives.

The positive periodic acid-Schiff reaction of colonic mucin, mixed tumor stroma and cartilage is completely inhibited by prior methylation for 96 hours at 58°C. and the reaction of gastric and small intestine mucins is only moderately reduced at this period. At room temperature no alteration is observed after methylation for 21 days. The positive reaction of human mast cell granules is abolished after 72 hours of treatment at 58°C. and is only moderately affected after treatment for 21 days at room temperature.
HISTOCHEMICAL DEMONSTRATION OF ASCORBIC ACID IN THE ADRENAL GLAND WITH A CRITICAL STUDY OF THE METHOD
Authors: ERÄNKÖ OLAVI
Published: April 2011

The silver nitrate technique for the histochemical demonstration of ascorbic acid has been applied to the adrenals of the rat. Whole glands, fresh-frozen and frozen-dried sections were treated with the silver reagent. Diffusibility of ascorbic acid in the adrenal tissue was studied in tissue disc preparations and with "section chromatography". The following conclusions were derived:

1) Concentrations of silver nitrate, acetic acid and alcohol in the reagent are not critical.

2) Light does not affect the results under the conditions of the present study.

3) Excess silver nitrate must be washed out before fixation in thiosulphate.

4) Ascorbic acid is in a highly diffusible form in the adrenal cortex.

5) The solvent penetrates adrenal tissue at a higher speed than silver nitrate.

6) The presence of silver granules in the zona glomerulosa and the medulla of glands immersed as a whole into the silver reagent is due to dislocation of ascorbic acid during the histochemical procedure.

7) Diffusion of ascorbic acid is reduced to the minimum by treating fresh-frozen or frozen-dried sections with the silver reagent. Silver granules are in such sections restricted to the fasciculata and reticularis.

8) Stimulation of the adrenal cortex with injections of adrenaline or ACTH cause a depletion of silver granules mainly in the lower parts of the fasciculata and the reticularis.

9) Histochemically demonstrable ascorbic acid is probably distributed evenly in the cell both in normal and in stimulated adrenals. This point requires further study.
SULPHATION TECHNIQUES IN HISTOCHEMISTRY WITH SPECIAL REFERENCE TO METACHROMASIA
Authors: KRAMER H. , WINDRUM G. M.
Published: April 2011

1. Methods are described for the sulphation of tissue carbohydrates in histological sections.

2. Evidence is produced to show that the various procedures described result in the formation of sulphuric acid esters of the carbohydrates.

3. The histochemical applications of the metachromatic staining reaction can be greatly extended by the use of these techniques, as neutral polysaccharides, non-sulphated acid mucopolysaccharides, and glycoproteins, which are ordinarily orthochromatic, are rendered metachromatic.
HISTOCHEMICAL DEMONSTRATION OF β-d-GLUCURONIDASE WITH A SYNTHETIC SUBSTRATE
Authors: SELIGMAN ARNOLD M. , TSOU KWAN-CHUNG , RUTENBURG SELMA H. , COHEN RICHARD B.
Published: April 2011

The synthesis of a substrate, 6-bromo-2-naphthyl β-d-glucopyruronoside, is described, and the procedure for the histochemical demonstrations of β-d-glucuronidase activity and experiments to determine the reliability of the results of localization of enzymatic activity are given. The results in normal tissues of the rat are presented. Greatest concentration of enzymatic activity was seen in liver, spleen, epithelium of gastrointestinal tract, uterus, thyroid, and in the white matter of the nervous system.
CYTOLOGICAL OBSERVATIONS OF THE EFFECT OF ENZYMES ON ACID-TREATED SPORES OF BACILLUS CEREUS
Authors: KEIGLER NORMA M. , SMITH ANDREW G.
Published: April 2011

Resting spores of Bacillus cereus were studied cytologically by means of a technique involving both acid and enzyme hydrolysis which was supplemented by selective staining and observation by both bright field and phase contrast microscopy. Enzymes were used singly and in two-enzyme sequences following acid hydrolysis.

The results show the artifactitious peripheral structure to be digested by the proteases pepsin and trypsin, and the central structure to be relatively resistant to such action.

The nucleases ribonuclease and desoxyribonuclease caused no dissolution of structures, but effected to different degrees a loss of basophilia in the peripheral structure. The former's lesser action led to the conclusion that some ribonucleic acid was associated with the spore's nuclear chromatin in a relatively acid resistant complex, thus, aligning the spore's nucleus with that of other plant and animal tissues in regard to the presence of a ribodesoxyribonucleoprotein complex.

Other than effecting the phase contrast phenomenon of reversal of contrast, two-enzyme sequences failed to result in striking increases in degradation and dissolution of the central and the peripheral structures.

Buffer and diluent effects on a substrate's subsequent susceptibility to enzyme action and to staining reactions were noted and were in accord with the finding-of other investigators. Osmium tetroxide fixation appeared to increase the subt strate's susceptibility to trypsin action. Magnesium sulfate was shown to inhibis pepsin activity.

Potassium permanganate was shown to be an effective mordant for increasing the basophilia of the central structure.
THE HISTOCHEMICAL SPECIFICITY OF CHOLINESTERASES
Authors: CHESSICK RICHARD D.
Published: April 2011

On the basis of a review of the biochemical and histochemical literature, all esterases are divided into the eserine-resistant esterases (aliesterases) and the eserine-sensitive esterases (cholinesterases). It has previously been demonstrated by histochemical study that a wide spectrum of enzymes exists among the aliesterases; the present study is concentrated on the cholinesterases. The myoneural junction in four species; man, dog, rat, and mouse, was studied by histochemical techniques utilizing both choline esters and non-choline esters as substrates, and under the influence of various physical and chemical agents. On the basis of this study, it is demonstrated that none of the biochemical classifications or `types' of cholinesterases can be defended, and that the cholinesterases of the various species and tissues should therefore be viewed as a family of enzymes, forming a spectrum with multiple overlapping and differences with respect to physical properties, effects of inhibitors and substrate specificities.
THE DESOXYRIBOSE NUCLEIC ACID (DNA) CONTENT OF HUMAN MONOCYTES AND THEIR DERIVATIVES DURING GIANT CELL FORMATION IN VITRO
Authors: GOLDSTEIN MILTON N.
Published: April 2011

1. The desoxyribose nucleic acid (DNA) content of individual cell nuclei in monocytes and their derivatives, multinucleated giant cells, has been determined by microspectrophotometry. These measurements were performed on Feulgen stained material.

2. Although most of the measurements of individual nuclei in cultures incubated from 4 to 138 hours were close to diploid value, there were variations in some nuclei well above and below this value.

3. The data indicated that DNA was being synthesized during cultivation.

4. Amitosis occurred in some of the nuclei of the multinucleated giant cells after these cells had formed by cell fusion.
ELECTRON MICROSCOPY OF BASOPHILIC COMPONENTS OF CYTOPLASM
Authors: PORTER KEITH R.
Published: April 2011

Electron microscopy of thinly spread cells grown in vitro has defined, as a component of the cytoplasm, a complex reticulum of strands and vesicles which has come to be called the endoplasmic reticulum. The component is limited by a membrane similar in thickness (ca. 80 Å) to the plasma membrane of the cell. It separates the content of the strands and vesicles from the general matrix of the cytoplasm and gives to the whole component the character of a finely divided vacuolar system. The elements of the system are frequently large enough to be resolved by light microscopy and can be shown to have an affinity for basic dyes. The system has therefore been identified as a basophilic component of the cytoplasm (25).

With the techniques now available for thin sectioning the same system of vesicles and interconnecting canaliculi can be demonstrated in cells fixed in situ and studies of such material have defined the system as a universally occurring component of the cytoplasm. In its structure it varies greatly from one cell type to another and even within a single kind of cell under different physiological states. In one form it is common for it to be made up of extraordinarily thin, flattened vesicles referred to as cisternae. In certain types of cells, particularly acinar cells of exocrine glands, these vesicles are organized in parallel arrays and such organizations have been shown to coincide with the distribution of the basophilic component.. Thus in thin sections as in cultured cells the reticulum is identified with the basophilic material.

In very recent electron microscopy of thin sections, Palade (16) has observed that a dense granular component of the cytoplasmic matrix is usually adjacent to the membrane surfaces of the reticulum. In rapidly growing cells where the basophilia is diffuse and where elements of the reticulum may not be prominent, granules of this latter character (100-300 Å diameter) are abundant and scattered throughout the cytoplasmic matrix. This suggests therefore that in the other instances where the intensely basophilic component and the endoplasmic reticulum coincide, the latter structure owes its basophile properties to the quantities of the granular material which associate preferentially with its membrane surfaces. This conclusion finds support in studies of cell fractions (1, 23) which have defined small cytoplasmic particles that are extraordinarily rich in ribonucleic acid. It is also supported by the observation that parts of the nucleolus consist of closely packed granules of similar size and density. It appears then that the basophilic component when it exists as a distinct and localized entity of the cytoplasm, consists of a concentration of vesicular and canalicular elements of the endoplasmic reticulum with small basophilic granules associated. Where the basophilia is diffuse it seems to be an expression of the diffuse distribution of the small granules.
THE EFFECTS OF RIBONUCLEASE ON THE RIBONUCLEIC ACID AND ENZYME ACTIVITIES OF MICROSOMES ISOLATED FROM RAT LIVER HOMOGENATES
Authors: NOVIKOFF ALEX B. , RYAN JEAN , PODBER ESTELLE
Published: April 2011

1. The RNA of isolated rat liver microsomes is readily accessible to ribonuclease digestion. About 85% of the RNA-ribose is removed by the nuclease; data in the literature suggest that the rest remains in smaller polynucleotides rather than RNA per se.

2. Digestion of microsomes with ribonuclease dissolved in glycyl-glycine buffer, pH 7.0, has no effect upon the activities of the enzymes studied: glucose-6-phosphatase, esterase, triacetic acid lactonase, adenosine-5'-phosphatase, acid phosphatase, adenosine triphosphatase, and succinoxidase.

3. Digestion with ribonuclease dissolved in water has no effect upon the acid phosphatase and adenosine-5'-phosphatase activities, while esterase, adenosine triphosphatase and succinoxidase activities are somewhat lower. Glucose-6-phosphatase activity is markedly reduced, as it is by incubation of microsomes with either water or buffer alone.
LOCALIZATION OF PHOSPHATASE ACTIVITIES IN THE RAT ANTERIOR PITUITARY GLAND
Authors: PELLETIER GEORGES , NOVIKOFF ALEX B.
Published: April 2011

All five known secretory cell types of the rat anterior pituitary gland display nucleoside diphosphatase (NDPase) activity throughout the endoplasmic reticulum (ER), including the nuclear envelope but not the specialized region of ER at the inner aspect of the Golgi apparatus known as GERL. The functions of the ER diphosphatase are currently unknown. However, speculations concerning its association with glucuronyl transferase may focus on the metabolic roles of the ER in pituitary cells other than those directly related to secretory protein transport. The gonadotrophs have been studied for thiamine pyrophosphatase and acid phosphatase activities as well as NDPase activity. The results suggest that the secretory granules of gonadotrophs arise from GERL and not from the inner element of the Golgi apparatus. The innermost Golgi element of this cell type shows NDPase and thiamine pyrophosphatase activities and appears to be composed, in part at least, of anastomosing tubules. Nucleoside phosphatase activity is also present at the surfaces of all five secretory cell types and between the cells and adjacent blood capillaries.
TRANSPORT ADENOSINE TRIPHOSPHATASE CYTOCHEMISTRY I. BIOCHEMICAL CHARACTERIZATION OF A CYTOCHEMICAL MEDIUM FOR THE ULTRASTRUCTURAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY IN THE AVIAN SALT GLAND
Authors: ERNST STEPHEN A.
Published: April 2011

The optimal kinetic parameters for the K-dependent, ouabain-sensitive hydrolysis of p-nitrophenyl phosphate by K-nitrophenyl phosphatase, under conditions closely approximating those employed for cytochemistry, were determined in the avian salt gland as a necessary prerequisite for the ultrastructural localization of the enzyme. The enzyme was characterized in 50-µ cryostat sections of paraformaldehyde-fixed tissue, incubated at room temperature in a medium containing 5 mM nitrophenyl phosphate, 10 mM MgCl2, 20 mM SrCl2 and 100 mM Tris-HCl buffer (pH 9.0), either with or without 10 mM KCl. For comparison, parallel assays were conducted in the absence of Sr, the heavy metal salt used to precipitate phosphate for cytochemistry. Enzymatic activity was determined by measuring spectrophotometrically the amount of nitrophenol hydrolyzed. In this system, Sr acts as a pure noncompetitive inhibitor of the enzyme, causing 50% inhibition at 3 mM and 87% at 20 mM. The Km for the enzyme is 4.5 mM. Sr (20 mM) causes an 8-fold reduction in the apparent affinity of the enzyme for Mg but has little effect on K affinity. The sensitivity of the enzyme to ouabain is decreased 50-fold in the presence of 20 mM Sr. The relationship of this enzymatic activity to Na-K-activated adenosine triphosphatase and the application of this defined medium to transport adenosine triphosphatase cytochemistry are discussed.
CRITIQUE ON THE K-PYROANTIMONATE METHOD FOR SEMIQUANTITATIVE ESTIMATION OF CATIONS IN CONJUNCTION WITH ELECTRON MICROSCOPY
Authors: KLEIN RICHARD L. , YEN SHYUE-SHONG , THURESON-KLEIN ÅSA
Published: April 2011

The histochemical method employing potassium pyroantimonate in conjunction with electron microscopy has been investigated using carefully controlled preparation techniques and very sensitive atomic absorption analysis of cations. A critique on the reliability and limitations of the method based on test tube and in vitro experiments is given. The method is sensitive to Ca++, Mg++ and Na+ at the 10–6, 10–5 and <10–2 M levels, respectively. Under defined conditions a linear ~l:l ratio of cation present to cation precipitated occurs above these levels. Approximate solubility products have been estimated. Under the test conditions, K+ does not precipitate as a pyroantimonate salt, and neither K+ nor OsO4 influences cation precipitaton at physiologic concentrations. Unbuffered, Tris-HCl-buffered and weakly buffered NaHCO3 media at pH 7.2-7.8 give statistically similar results with Na+ precipitation. The pyroantimonate ion can compete with chelators, ethylenedinitrilotetraacetic acid and ethylene glycol bis-N, N'-tetraacetic acid, for divalent cations when employed simultaneously. These chelators effectively remove Ca++ but not Mg++ from embryonic myocardium, and their effects on Na+ and K+ balance are not marked if employed for relatively short periods. Electron micrographic examples of cation precipitates are given in support of certain findings. A brief discussion of the significance of pyroantimonate grain size, the discrepancy between the ratio of intra- and extracellular precipitates and guidelines for the use of the method are included.
ACETYLCHOLINESTERASE AT THE MYONEURAL JUNCTION: CYTOCHEMICAL ULTRASTRUCTURE AND SOME BIOCHEMICAL CONSIDERATIONS
Authors: FRIEDENBERG ROBERT M. , SELIGMAN ARNOLD M.
Published: April 2011

Recent studies of acetylcholinesterase (AChE) and related enzyme systems at the myoneural junction are reviewed primarily from the ultrastructural cytochemical point of view. The four most prevalent methods used to determine localization of AChE ultrastructurally are: (a) a hexazotized pararosaniline diazo coupling method, (b) a thiolacetic acid method and modifications, (c) a thiocholine method and modifications and (d) osmiophilic thiolester methods. After an initial discussion of key questions germaine to the subject matter and a short summary to introduce ultrastructural considerations, the principles upon which the above four groups of methods are based are described with a critical evaluation of their advantages and disadvantages. Such characteristics as specificity, localization, penetration, ascertaining bound and unbound enzymes, dangers of nonenzymatic reactions and contrast in the electron microscope from the primary reaction product are detailed and compared for each method. A limited number of biochemically related topics are also considered which may have a bearing on cytochemical ultrastructural results. Of immediate interest are recent studies of isolation, purification and assay of AChE along with quantitation and a comparison of findings in animals and man. Other enzyme systems (other than AChE) at the motor end plate are also surveyed. The characteristics of the acetylcholine system and other neurohumoral transmitter systems are noted in some detail. Other less closely related topics included in this review but still of great interest are: localization in neural and nonneural tissue; the use of inhibitors, antibiotics and radiochemical methods; localization and isolation of receptor sites; use of brain synaptosomes; and two general theories linking neurohumoral transmission concepts and the metabolism of phosphorus compounds to the localization of AChE. No attempt has been made to be complete with these biochemical considerations. Rather, the reader is led into the literature as a first step in relating the cytochemical findings to biochemical and physiologic mechanisms.