bluuebunny/crossref_metadata_2025 huggingface dataset:
MICROSCOPIC HISTOCHEMICAL DEMONSTRATION OF STEROID-3β-OL DEHYDROGENASE IN TISSUE SECTIONS
Authors: WATTENBERG LEE W.
Published: April 2011

A procedure for the microscopic histochemical demonstration of steroid-3β-ol dehydrogenase has been described. In agreement with reported quantitative biochemical data, positive reactions have been obtained in the adrenal cortex, corpus luteum, placenta, and interstitial cells of the testis. In addition, a strongly positive reaction is obtained in the interstitial cells of the rodent ovary.
A SEMI-MICRO METHOD FOR THE DETERMINATION OF CRYSTAL VIOLET SORBED TO BIOLOGICAL MATERIAL
Authors: FISCHER ROLAND , ZALESCHUK JERRY
Published: April 2011

A semi-micro method is reported to measure the degree of Gram positive behaviour, i.e. the amount of crystal violet sorbed by various proteinous materials. Part of the role supposedly played by "loosely bound" iodine as well as that by covalently bound iodine in the crystal violet uptake of intact wool (Gram negative) and alkali-degraded wool (Gram positive) has been determined. The influence of reversing the consecutive steps of staining and iodination on dye-uptake has been measured for wools. The iodine uptake prior to and after staining, as well as the dye-uptake prior to iodination, have been determined for the two types of wool as well as for Escherichia coli and Leuconostoc mesenteroides respectively.

The dye-uptake of wools and of bacteria was also measured after treatment with 1-fluor-2,4-dinitrobenzene.

The identical composition in non-sulphur containing amino acids of intact wool and alkalidegraded wool—prior to and after treatment with 1-fluor-2,4-dinitrobenzene—has been investigated by subjecting the wools to hydrolysis and one- as well as two-dimensional paper chromatography.

Data are reported as to the crystal violet uptake prior to and after iodination of muscle and nerve-fibres, nail, ovalbumin, β-glucuronidase and pepsin.

The theoretical implications of these and other data are discussed and attention is drawn to the manyfold aspects of the problem of Gram staining behaviour of proteins.
CHARACTERIZATION OF SCHIFF-POSITIVE SUBSTANCES IN ELASTIC FIBERS
Authors: LABELLA FRANK S.
Published: April 2011

Schiff-positive substances in clastic fibers have been separated on the basis of differences in reactivity with the reagent.

Alkali-treated ligamentum nuchae powder contains a cerebroside which is removed by chloroform or pyridine. Also present are a plasmalogen-like compound which depends upon its unsaturated component for its reactivity, and a substance whose Schiff positivity is negated by prior treatment with iodine; both reactants are resistant to extraction techniques. Mercuric chloride or enzymatic hydrolysis releases an unsaturated fatty material, probably the aldehydic component of the clastin-bound plasmalogen-like compound. Elastin powder which has been treated with phenylhydrazine, iodine and bromine still gives a positive reaction with fuchsin-sulfurous acid.

Rat aortic clastica, like ligamentum nuchac, contains a non-extractable plasmalogen like compound whose reactivity with Schiff's reagent depends Upon unsaturation in the molecule. Staining intensity is reduced by prior chloroform extraction, suggesting a glycolipid as was demonstrated for ligamentum nuchae.
QUANTITATIVE DETERMINATION OF CATIONIC DYEBINDING IN CONNECTIVE TISSUE
Authors: BALAZS ENDRE A. , SZIRMAI JOHN A.
Published: April 2011

Mucoid layer of the rooster comb and cartilage of the nasal septum of steers were used to measure the binding of cationic dyes by unfixed frozen sections. The amount of dye bound was calculated from the decrease of the optical density of the dyebath after equilibrium had been reached between the dyebath and the tissue at the saturation level. Various factors influencing this reaction, such as the tissue-dye ratio, the dyebath concentration, the thickness of the tissue sections and mechanical agitation, have been studied. In order to quantitatively determine the dyebinding, certain conditions must be fulfilled. The dyebath concentration must be high enough to be well in excess of all available binding sites of the tissue. The precipitate formed and the tissue fragments must be removed prior to optical density measurement. The electrolyte concentration of the dyebath must be kept below a certain level. The dyebath should not contain colored substances other than the free dye. Under these conditions, the amount of dye bound by azure A and other cationic dyes is constant for a given tissue, depending only on the pH of the dyebath. Electrostatic interaction between the cationic dye and the anionic sites of the tissue is primarily responsible for dyebinding. The quantitative determination of dyebinding is a measure of the free anionic sites of a tissue.
FIXATION IN CYTOCHEMISTRY AND ELECTRON-MICROSCOPY
Authors: BAKER JOHN H.
Published: April 2011

The purpose of the address is to point out the need for a fuller study of fixation for electron-microscopy, and especially to urge that cytochemists should interest themselves more actively in the interpretation of electron-micrographs. In some cases there is strong reason to suppose that the detailed structure seen in micrographs represents rather well the structure present in life. Certain globules in the neurones of the common snail, Helix aspersa, are carefully considered in this connection. In other cases, on the contrary, it is probable that the detailed structure seen in electron-micrographs does not represent the living condition. The structure is better interpreted as a reaction-product between the fixative used and what was present in life. In many cases, much of the detail is likely to be caused by the unmasking of hydrophil lipids from lipoprotein complexes. Such lipids, after unmasking, would be likely to arrange themselves in double membranes. The different ways in which fixatives may react with lipoproteins ate briefly described.
A NOTE ON CHOLINESTERASE ACTIVITY IN POST-MORTEM TISSUES
Authors: MOORE ELIZABETH J. , PETTY CHARLES S.
Published: April 2011

Cholinesterase activity at the myoneural junction in human intercostal muscle was demonstrated histochemically. Activity was present in refrigerated muscle up to 157 days and for a similar time in tissues removed from bodies within 24 hours after embalming. The duration of cholinesterase activity in muscle treated with various fixatives was also investigated.
ENZYME KINETICS IN A HISTOCHEMICAL SYSTEM
Authors: BENDITT EARL P. , ARASE MARGARET
Published: April 2011

Modification of the Lineweaver and Burk method of handling the chemical kinetics of an enzyme system has been made to suit the histochemical conditions. Thus if the reaction is allowed to run to some fixed quantity of end point 1/v becomes proportion to the time necessary to reach this end point. In practice serial dilutions of substrate and serial increments in time of incubation are set up. Slides are then separated under the microscope into groups of equal intensity. A plot of 1/[S] v.s. t fits a straight line as expected. Competition between two substrates, one chromogenic the other non-chromogenic, can be demonstrated. One source of deviation from the theoretical due to spontaneous hydrolysis of substrate has been recognized and can be accounted for.
ON THE EXPERIMENTAL DISCHARGE OF THE GASTRIC AND INTESTINAL ARGENTAFFIN CELLS
Authors: LILLIE R. D. , WEISSBACH H. , GLENNER G. G.
Published: April 2011

Reserpine discharges the azo positive reducing substance from both gastric and intestinal basal granular cells in the guinea pig. Regeneration was slower than in previous reports and apparently more rapid in the duodenum than in the stomach or ileum.

The numbers of cells demonstrated in the normal duodenum and ileum respectively, as well as in the duodenum and ileum of the 7 day post reserpine animal, do not correspond particularly closely with the 5-hydroxytryptamine assays.

Physostigmine and ephedrine failed to discharge the reactive material from basal granular cells of the guinea pig stomach and intestine.

Argyrophil cells persist throughout the reserpine discharge period, during which argentaffin, azo positive basal granular cells are not demonstrable.
THE DISTRIBUTION OF ESTERASES IN MOUSE TISSUES
Authors: MARKERT CLEMENT L. , HUNTER ROBERT L.
Published: April 2011

The esterases contained in 32 mouse tissues and organs have been analyzed using the zymogram technique. More than ten different esterases have been resolved and partly characterized by their substrate and inhibition specificities. The esterases compose a family of distinct enzymes with overlapping specificities, and the total activity in any tissue is the sum of the activities of the particular esterases in that tissue. Characteristic similarities and differences among the esterase patterns from different tissues were noted and discussed. The G band is apparently a cholinesterase as shown by its sensitivity to inhibition by eserine at 10–4 M. The small effect of age, sex, genotype, and nutrition upon the esterase pattern is contrasted with the striking changes occurring during the differentiation of organs. The greatest change not associated with differentiation was observed in blood after 5 days of starvation.

Fractionation of liver homogenates by centrifugation showed esterase to be concentrated primarily in the microsome fraction. Measurements were made of the quantitative distribution of esterase activity among the electrophoretically resolved bands from each of the centrifugal fractions of liver homogenates. The pattern of esterases was very similar in each of the centrifugal fractions.
NEW HISTOCHEMICAL TECHNIQUES FOR THE DEMONSTRATION OF TISSUE OXIDASE (CYTOCHROME OXIDASE)
Authors: BURSTONE M. S.
Published: April 2011

A group of complex naphthols, as well as open chain and cyclic methylene compounds, were found to be useful new reagents for the demonstration of tissue oxidase in conjunction with N-phenyl-p-phenylenediamine. The methylene compounds constitute a new class of histochemical reagents.

N-alkyl amines (e.g. N,N-dimethyl arylamines) are toxic to the enzyme and are to be avoided. Complex amines are inhibitory and are likewise not recommended.

As with the original "Nadi" technique, the reaction was inhibited by cyanide, azide, and sulfide. The distribution of staining in tissues was essentially identical with all reagents. Although the reaction was augmented by cytochrome c, this expensive reagent is not neeeded with the present techniques.

The indoaniline and azamethine dyes produced are highly insoluble and result in permanent or relatively permanent preparations. Most of the dyes are capable of complexing with mercury, molybdenum, cobalt, nickel, cadmium, lead, uranium, and iron, and thus may have potential applications in electron microscopy.

Among the naphthols 1-hydroxy-2-naphthoic acid (IV), 1-hydroxy-2-acetonaphthone (V), and a derivative of 1-hydroxy-2-naphthanilide (IX) are very useful couplers. Useful methylene compounds are exemplified by 1-phenyl-3(m-nitrobenzamidopyrazolone) (XII), naphthol AS-LG (XVIII), and naphthol AS-L3G (XX). Use of methylene compounds results in highly stable incubating solutions. All substrates are commercially available and inexpensive.

Unfixed frozen sections mounded on glass slides were used. Considerable caution must be exercised in attempts to localize oxidative enzyme activity at the cytologiocal level in unfixed frozen sections. This is due to the disruption of mitochondria and other cytological constituents which may occur during freezing and thawing. An alternative technique was evolved. This consisted of incubating fresh tissue slices, freezedrying them, followed by embedding in paraffin. Sections were then cut, dewaxed, and mounted.

Preliminary results indicate that tissue oxidase may also be demonstrated using a mixture of amines (e.g., N-phenyl-p-phenylenediamine and 2-methoxy-N-phenylenediamine) which are capable of forming complex azine dyes. The possibility of demonstrating other enzymes of the oxidase dehydrogenase complex warrants further investigation.
STUDIES OF SULPHUR, PHOSPHORUS AND SOME METALS IN SKIN FOLLICLES
Authors: RYDER M. L.
Published: April 2011

Radioactive sulphur could be detected rat and mouse follicles sooner after injection than in the lamb, and this may be associated with the faster growth rate of hair in the rat and mouse.

It was not possible to detect copper in follicles, although zinc was detected and found to be concentrated in the unkeratinised part of the fibre. Potassium was found in the outer sheath and in the unkeratinised part of the fibre, as well as in the stratum corneum.

Labelled phosphorus was detected mainly in the bulb, with smaller amounts in the unkeratinised part of the fibre.
THE DISTRIBUTION OF DEOXYRIBONUCLEASE IN NORMAL, CIRRHOTIC AND NEOPLASTIC RAT LIVERS
Authors: DAOUST R. , CANTERO A.
Published: April 2011

The distribution of deoxyribonuclease in normal, cirrhotic and neoplastic rat livers was investigated histochemically using the gelatine-DNA film method.

The bile duct cells and connective tissue elements which are present in abnormal amounts in cirrhotic liver contain little DNAase activity compared with parenchymal tissue. The distribution pattern of DNAase is relatively uniform in normal liver parenchyma but becomes heterogeneous in the parenchyma of cirrhotic and neoplastic tissues. Groups of parenchymal cells in cirrhotic liver and the hepatoma cells in general appear devoid of DNAase activity. The necrotic areas of tissues, on the other hand, show intense enzyme activity.
ALDEHYDE-FUCHSIN FOR PITUITARY CYTOCHEMISTRY
Authors: ELFTMAN HERBERT
Published: April 2011

The component of the aldehyde-fuchsin stain which is useful for pituitary staining requires more delicate manipulation than the elastic tissue component. Manufacture of the stain for pituitary purposes may he shortened to 26 hours by subjecting a 0.5% basic fuchsin solution in 70% ethanol, 0.75% paraldehyde and 1.25% HCl to incubation at 37°C. The pH of this solution precludes background staining, allowing aldehyde-fuchsin to be followed by the periodic acid-Schiff routine. Aldehyde fuchsin may be rejuvenated by adding basic fuchsin in alcoholic solution.

Oxidation of pituitary tissue allows aldehyde-fuchsin to stain not only thyrotrophs but gonadotrophs as well and accentuates the staining of the hypothalamic hormones and the Golgi bodies. Since some fixatives, such as dichromates, oxidize tissue, assay of the significance of aldehyde-fuchsin staining involves awareness of the influence of the particular fixative employed.

Spectrophotometric analysis of young and old aldehyde-fuchsin stains supports the assumption that staining of the pituitary thyrotrophs may involve other constituents of the stain complex than those relied on for elastic tissue visualization.
SPECIFICITY OF LABELLED THYMIDINE AS A DEOXYRIBONUCLEIC ACID PRECURSOR IN RADIOAUTOGRAPHY
Authors: AMANO M. , MESSIER B. , LEBLOND C. P.
Published: April 2011

A simple method has been devised for the extraction of deoxyribonucleic acid from histological sections by means of the enzyme DNAse without significant loss of ribonucleic acid.

Removal of deoxyribonucleic acid by this method completely eliminated the radioautographic reactions observed over nuclei after injection of thymidine-H3. It is concluded that labelled thymidine is an adequate tracer of newly formed deoxyribonucleic acid in radioautographs.
LIMITATIONS IN THE HISTOCHEMICAL DEMONSTRATION OF PYRIDINE NUCLEOTIDE-LINKED DEHYDROGENASES ("NOTHING DEHYDROGENASE")
Authors: ZIMMERMANN H. , PEARSE A. G. E.
Published: April 2011

1. The ability of SH groups in well-dialyzed protein fractions to reduce exogenous pyridine nucleotides has been described in the biochemical literature.

2. This effect has been shown to be the cause of the positive histochemical result obtained when fresh tissue sections are incubated at alkaline pH levels with oxidized pyridine nucleotides (DPN or TPN) and tetrazolium salts, in the absence of any substrate.

3. The effect is pH-dependent, commencing at about pH 7.0 and rising to a maximum between pH 8.0 and 9.0. It is abolished by low concentrations of sulphydryl inhibitors or by heating the tissues.

4. The specific diaphorases are unaffected by sulphydryl inhibitors and these are presumed to transfer hydrogen and electrons from the reduced pyridine nucleotides (DPNH, TPNH) to the tetrazolium salt.

5. The development of histochemical methods for specific pyridine nucleotide-linked dehydrogenases is hindered by the activity described, to which Racker's convenient term "nothing dehydrogenase" has been applied.
TETRAZOLIUM STUDIES ON THE RETINA: II. SUBSTRATE DEPENDENT PATTERNS
Authors: COGAN DAVID G. , KUWABARA TOICHIRO
Published: April 2011

Unsectioned retinas were incubated in a medium consisting of buffer, blue tetrazolium and one of the following substrates: lactate, lactate-DPN, diphosphopyridine nucleotide (DPN), DPNH, and succinate. Control retinas incubated without substrate showed only an occasional reduction of tetrazolium and this was so slight as to be inconsequential.

With lactate as the sole substrate no appreciable reduction of tetrazolium occurred at neutral pH but when DPN was also added a constant pattern of precipitation occurred that was qualitatively the same in all species. This pattern, associated with lactate-DPN substrate, was characterized by precipitation of tetrazolium in ganglion cells, Müller's fibers, and other glial cells. It was particularly obvious in human retinas and most particularly in pathologic retinas which were accompanied by gliosis. Also characteristic was the absence of activity in the ellipsoids of the rods and cones.

The lactate-DPN activity was somewhat enhanced by incubation anaerobically or with cyanide. It was completely inhibited by p-chloromercuribenzoate or by borate. It was not affected by prior blinding of the animal with iodoacetate, and section of the optic nerve affected it only by abolishing the activity in the ganglion cells.

When DPNH was used as a substrate the reduction of tetrazolium followed the same pattern. The inference is that lactate contributes the electron to DPNH and that tetrazolium reduction is effected through diaphorase. On the other hand lactate activity occurs at an alkaline pH (8.8-9.2) without the addition of DPN.

In contrast to the lactate-DPN system, succinate substrates showed predominant activity in the ellipsoids of the rods and cones and in inner portion of the retina unassociated with glial cells or ganglion cells. It was approximately equal in all species. The succinate activity was greatly decreased in pathologic retinas associated with gliosis. It was inhibited by cyanide and malonate but not by borate. Section of the optic nerve caused no appreciable alteration but prior blinding of the animal with iodoacetic acid showed a loss of activity from the rods and cones and a significant increase in activity in the inner retinal layers.

It is concluded that tetrazolium precipitation in the retina with lactate-DPN as a substrate is most marked in the glial cells and ganglion cells whereas that with succinate as a substrate is most marked in the nerve fibers and in the ellipsoids of the rods and cones.
THE ACTIVITY OF SUCCINIC DEHYDROGENASE IN THE EXPERIMENTAL EPENDYMOMA OF C3H MICE
Authors: OGAWA KAZUO , ZIMMERMAN H. M.
Published: April 2011

1. The activity of succinic dehydrogenase in the transplantable ependymoma induced with methylcholanthrene in C3H mice was studied by both biochemical (QO2) and histochemical (using nitroneotetrazolium chloride) methods under aerobic conditions. The mitochondria were identified by Mallory's method in fixed tissue.

2. Succinic dehydrogenase activity in the ependymoma was much lower than in normal cerebrum.

3. In the normal cerebrum as well as in ependymoma there was close parallelism between the number, distribution and localization of mitochondria and succinic dehydrogenase activity.

4. In the normal cerebrum, neurons revealed the highest succinic dehydrogenase activity, followed by choroidal epithelium and ependymal cells. Formazan granules were scattered throughout the neuropil. Oligodendrocytes, particularly satellites, showed moderate activity. Occasionally very low activity was present in astroglia.

5. Ependymoma cells contained a smaller number of formazan granules than average normal cerebrum, and the mitochondrial dots, rods and filaments in the ependymoma cells were smaller, finer and fewer. In addition, ependymoma cells in mitosis appeared to have lower succinic dehydrogenase activity and to be poorer in mitochondria than cells in the interphase.

6. It is postulated that the low enzymatic activity observed in ependymoma homogenates is related mainly to the scarcity of mitochondria and in part to the high incidence of mitotic cells.
ENZYMES CATALYZING SEQUENTIAL REACTIONS IN MOUSE BRAIN AND LIVER SUPERNATANT FRACTIONS: I. DIFFERENTIAL USE OF JANUS GREEN B AND PHENAZINE METHOSULFATE
Authors: DU BUY HERMAN G. , SHOWACRE JANE
Published: April 2011

(1) Janus green B and phenazine methosulfate were used to determine the presence of single enzymes, or enzyme systems, which catalyze sequential reactions in brain and liver supernatants.

(2) Spectrophotometric studies on the reduction of Janus green B by supernatants demonstrated the absence of functional flavoprotein in brain supernatants and their presence in liver supernatants, corroborating results obtained by manometry.

(3) Brain supernatants contained at least 0.5 microgram of total flavin per ml equivalent; liver supernatant contained about 7 micrograms per ml equivalent. Therefore, the inability of brain supernatant to react with Janus green B was due not to the absence of riboflavin or flavin-adenine dinucleotide, but to the absence of a functional flavoprotein.

(4) The absence of a functional flavoprotein did not eliminate the activity of the glycolytic enzyme system in brain supernatants as measured by substrate-dependent lactate accumulation.

(5) A flavin inhibitor, acriflavin, did not significantly block glycolysis in brain or liver supernatants, either in the presence or absence of Janus green B.

(6) There was no evidence for the presence of a soluble flavin inhibitor in brain supernatants.

(7) Reduced diphosphopyridine nucleotide, used as substrate, was oxidized endogenously at a slow rate by both supernatants. This rate was increased by Janus green B in liver but not in brain supernatants.

(8) In the presence of pyruvate and Janus green B, both supernatants rapidly oxidized reduced diphosphopyridine nucleotide, but the dye remained oxidized. This indicates that the flavindye-oxygen electron transport chain was inoperative in the presence of the lactic dehydrogenase-pyruvate system of both supernatants.

(9) Where the absence of functional flavoprotein prevented the use of Janus green B for the demonstration of enzyme systems, phenazine methosulfate was used. Oxygen uptake in the presence of this dye was a measure of reduced diphosphopyridine nucleotide formed. Thus, it was a measure of the activity of enzymes or enzyme systems, of which diphosphopyridine nucleotide-requiring dehydrogenases are components.

(10) By supplying phenazine methosulfate as terminal electron transport agent, both supernatants were shown to contain enzyme systems which metabolized fructose diphosphate, lactate, and malate.

(11) The results are discussed with reference to the thesis that the enzyme systems occurring in supernatants represent fractions of those retained by the corresponding mitochondria.
A RELATION BETWEEN TOTAL GLYCOGEN CONTENT OF OX MYOCARDIUM AND ITS HISTOCHEMICAL DEMONSTRATION
Authors: KUGLER J. H. , WILKINSON W. J. C.
Published: April 2011

Glycogen has been determined chemically in ox myocardium by the anthrone method and histochemically by Gomori's hexamine silver technique. It is concluded from the results that there must be present in the tissue a minimal quantity of glycogen before it can be demonstrated histochemically. A value of 185 µg/100 mg tissue (wet weight) is presented as this quantity.
OBSERVATIONS ON THE DIAZOTIZATION-COUPLING REACTION FOR THE HISTOCHEMICAL DEMONSTRATION OF TYROSINE: METAL CHELATION AND FORMAZAN VARIANTS
Authors: GLENNER G. G. , LILLIE R. D.
Published: April 2011

1. With the finding of the photosensitivity of protein-bound diazotyrosin, a reproducible and sensitive reaction for tyrosine and certain other phenolic compounds in tissue sections based on diazotization and coupling is presented.

2. The specificity of the diazotization-coupling reaction for protein-bound tyrosine among the amino acids is established and the histochemical localization of tyrosine-containing protein in numerous tissues is described.

3. A modification producing a formazan product is presented, which, however, is too insensitive for exacting histochemical use. A modification producing o,o’-dihydroxyazo compounds, which give excellent histochemical localization and are capable of metal chelation, is described, and the possible use of the uranyl chelate for the demonstration of tyrosine by electron microscopy is suggested.
MICROPHOTOMETRIC ANALYSIS OF THE CYTOCHEMICAL MILLON REACTION
Authors: RASCH ELLEN , SWIFT HEWSON
Published: April 2011

The cytochemical Millon reaction has been studied with test protein systems and with several plant and animal tissues. Absorption curves of the Millon chromophores have been analyzed and compared with natural ultraviolet protein absorption. The findings indicate the validity of applying the Millon reaction as a quantitative cytochemical method in studies of the nucleoprotein complexes of cells, but only when proper corrections for non-specific light loss and other variables have been applied. Under these conditions, a roughly linear relationship was found between natural ultraviolet protein absorption at 280 mµ and the intensity of the Millon chromophore at 500 mµ. A scheme of intermediate products and competing reactions coincident to formation of Millon chromophores has been proposed, based on changes in absorption spectra during color development in test protein systems.
ELASTOLYTIC EFFECT OF PEPSIN
Authors: FISHER EDWIN R. , ROSENTHAL T. B. , LANSING ALBERT I.
Published: April 2011

Pepsin exhibits an elastolytic effect on elastic fibers of human skin and aorta in tissue sections fixed in alcohol or formalin which precedes its well recognized collagenolytic action in such preparations. Elastin obtained from ligamentum nuchae is similarly solubilized by pepsin in vitro. Its action has been estimated to be approximately one-eighth that of several elastase preparations tested.

The susceptibility of elastic fibers and elastin to peptic digestion suggests that they possess the aromatic acid residues tyrosine and phenylalanine in combination with glutamic acid. Carboxyl groups do not appear essential for this peptic activity since prolonged methylation failed to inhibit the elastolytic effect of pepsin on elastic fibers in tissue sections.
A SIMPLIFIED METHOD FOR DEMONSTRATION OF CALCIUM IN SPODOGRAMS
Authors: GODLEWSKI H. G.
Published: April 2011

A simple atomiser is used in order to spray the sulphuric acid over the spodogram. The formed crystals of gypsum prove the presence of calcium in particular tissue structures. The procedure avoids the use of micromanipulator applied in the original method. Gypsum crystals have been demonstrated in the wall of the hamster cheek pouch, the developing tooth of new-born hamster and in the sclerotic human aorta.
HISTOCHEMICAL DEMONSTRATION OF UNSATURATED LIPIDS BY BROMINE SILVER METHOD
Authors: MUKHERJI MRITYUNJOY , DEB CHANDICHARAN , SEN PARIMAL BIKAS
Published: April 2011

A method of demonstration of unsaturated lipids in tissue sections has been described.

Formol fixed frozen sections are exposed to bromine vapour. It is then allowed to react with an alcoholic or ammoniacal solution of silver nitrate, the silver salt being finally reduced to metallic state by reducing agent.

Evidences have been put forward to prove the superiority of bromine over iodine treatment.

If properly controlled, the method appears to be quite specific.
ESTERASE ACTIVITY IN LEUKOCYTES DEMONSTRATED BY THE USE OF NAPHTHOL AS-D CHLOROACETATE SUBSTRATE
Authors: MOLONEY WILLIAM C. , MCPHERSON KENNETH , FLIEGELMAN LILA
Published: April 2011

1. Human myeloid cells in the peripheral blood and bone marrow contain a very strong esterase activity which can be demonstrated histochemically by the use of naphthol AS-D chloroacetate as substrate.

2. As first pointed out by Gomori, this esterase activity is found not only in segmented but also immature myeloid cells. However, stem cells, cosinophilic and basophilic granulocytes, lymphocytes, monocytes, plasma cells, megakaryocytes, adult red cells and red cell precursors do not stain with this method.

3. Histocytes and mast cells are strongly positive for this esterase activity.

4. No differences in esterase activity have been noted thus far between neutrophilic leukocytes in normal individuals and in patients with chronic myclogenous leukemia or other diseases.

5. The leukocyte esterase activity is abolished by various well known inhibiting agents. Activity in young myeloid cells in non-leukemic bone marrow smears and in the peripheral blood of patients with myeloid leukemia is inhibited less than that in adult segmented neutrophils.

6. The functions of this esterase in leukocytes are unknown, but further studies of this enzyme seem warranted.
SOME OBSERVATIONS OF THE REACTION OF SCHIFF REAGENT WITH ALDEHYDES
Authors: BARKA TIBOR , ORNSTEIN LEONARD
Published: April 2011

From the reaction mixture of Schiff reagent and formaldehyde 3 to 6 (sometimes even 7) compounds can be separated on paper chromatogram. The Rf values of these compounds range from .09 to .74 and the absorption maxima from 535 to 574 mµ. The formation of these compounds is influenced by at least two factors: the SO2-content of the Schiff reagent and the molar ratio of aldehyde to Schiff reagent. Saturation of the Schiff reagent with SO2 influences the hue of Feulgen stained nuclei and reduces the variation in Feulgen microspectrophotometry.
REGIONAL DIFFERENCES IN ACID PHOSPHATASE AND TWEEN ESTERASE ACTIVITY IN THE EPIDERMIS OF THE CHIMPANZEE AND THE MACAQUE
Authors: MORETTI GIUSEPPI , ADACHI KENJI , ELLIS RICHARD A.
Published: April 2011

Using Semi-quantitative and photometric techniques, regional variations in the activity of acid phosphatase and Tween esterase have been demonstrated in the epidermis of the chimpanzee and macaque. The observed differences cannot be directly correlated with regional changes in the morphology of the epidermis. It is suggested that similar variability in a multiplicity of enzymes may account for the structural differences, and obscure specific correlations with the two enzymes we have studied.
OSMIUM TETROXIDE AND THE MARCHI METHOD: REACTIONS WITH POLAR AND NON-POLAR LIPIDS, PROTEIN AND POLYSACCHARIDE
Authors: ADAMS C. W. M.
Published: April 2011

1. The reactions of tissues with OsO4 and the Swank-Davenport KClO3-OsO4 (Marchi) reagent have been studied by blockade of the reducing groups of unsaturated lipid, protein and polysaccharide. Only blockade of the —CH=CH— bond prevented osmiophilia after brief staining (18 hr.).

2. Tissue-protein is neither stained nor demonstrably impregnated after brief treatment with OsO4.

3. Chromatographic separation of degenerating myelin revealed that the Marchi-substance is eluted in the esterified cholesterol fraction.

4. Unsaturated lipid but not protein or polysaccharide are stained by brief treatment with OsO4. The Marchi reaction distinguishes hydrophilic from hydrophobic unsaturated lipid.
"pH SIGNATURE" OF ADENOCARCINOMA OF THE COLON IN MAN
Authors: FORAKER ALVAN G.
Published: April 2011

Sections from ten cases of adenocarcinoma of the human colon were stained with methylene blue and with light green SF in a range of pH. Staining intensity of various cellular structures in normal and neoplastic mucosa was recorded on an arbitrary scale. This procedure is said to assist in characterization of proteins. Nuclear structures stained more intensely with methylene blue in alkaline ranges. Mitotic figures in carcinoma retained affinity for methylene blue in relatively acid ranges when other nuclear structures stained weakly. Cytoplasmic staining with light green increased in acid ranges. Only mucin showed some metachromasia. None of the findings sharply differentiated between normal and carcinoma cells.
HISTOLOGY AND CYTOCHEMISTRY OF HUMAN SKIN. XX. THE DISTRIBUTION OF MONOAMINE OXIDASE
Authors: YASUDA KENJIRO , MONTAGNA WILLIAM
Published: April 2011

1. Using the method of Glenner et al. (8), we have demonstrate abundant monoamine oxidase in human skin.

2. In the epidermis, the reaction occurs in the cells of the malpighian layer. In all hair follicles, regardless of their states of activity, the wall of the pilosebaceous canal is intensely reactive. In growing follicles, the cells of the outer root sheath contain moderate enzyme activity particularly concentrated in the cells at the periphery, in the region above the bulb; small reactive granules are sparsely distributed in the cells of the dermal papilla. In quiescent follicles, there is moderate activity in the cells of the epidermal sac; the hair germ and the dermal papilla have a scant reaction. In the sebaceous glands, the duct and the undifferentiated cells at the periphery of the acini have a very strong reaction. Both apocrine and eccrine glands show strong enzyme activity.

3. Many cutaneous nerves contain monoamine oxidase. Reactive nerves form plexuses just beneath the epidermis, at the level of the sebaceous gland, and midway along the depth of the hair follicle; reactive nerves are also found around both eccrine and apocrine glands. Eccrine glands are surrounded by a richer bed of nerves than the apocrine glands are. The nerves to the arteries also contain the enzyme.

4. The localization of monoamine oxidase in human skin resembles that of succinic dehydrogenase; the cutaneous nerve fibers and the myoepithelial cells, which contain no succinic dehydrogenase, do, however, contain monoamine oxidase.

5. Many cutaneous nerve fibers, sensory and motor, seem to be reactive for both monoamine oxidase and cholinesterase, We do not know what this means in terms of physiology.
THE DISTRIBUTION OF CHOLINESTERASE IN THE CUTANEOUS RECEPTOR ORGANS, ESPECIALLY TOUCH CORPUSCLES OF THE HUMAN FINGER
Authors: CAUNA NIKOLAJS
Published: April 2011

1. Cutaneous receptor organs of the human digital skin have been investigated for cholinesterase activity by a modification of the histochemical technique of Koelle and Friedenwald, and the findings have been analyzed in relation to the structure of the receptors.

2. It was found that in Meissner's corpuscles the nerves and their sheaths were negative, but the plasma membranes of the laminar cells and the intercellular substance gave strong positive reactions with either acetyl or butyryl substrates.

3. In Merkel's corpuscles, nerve endings and the associated Merkel's cells were negative, but the preterminal medullated nerve fibres gave positive reactions with the butyryl substrate.

4. In hairs, the terminal nerve fibres surrounding the epithelial sheaths gave positive reactions with both substrates.

5. Free nerve endings were negative but the cells related to the endings gave a positive reaction with both substrates.

6. Capillaries and sebaceous glands were negative, but adventitia of larger blood vessels gave positive reactions with the butyryl substrate.

7. The significance of the findings is discussed and a suggestion is made that both acetylcholine and its enzyme may constitute active agents in the synaptic transmission of a nervous impulse in Meissner's corpuscle.
CHOLINESTERASE IN THE CRUSTACEAN MUSCLE RECEPTOR ORGAN
Authors: MAYNARD E. A. , MAYNARD D. M.
Published: April 2011

The muscle receptor organs of Homarus americanus were examined histochemically for cholinesterase, using the thiocholine method of Koelle. Cholinesterase occurs in the sensory neurons and in motor and accessory fibers, but its highest concentration is reached in the interelated regions of the muscle receptor organs. The somatic cytoplasm of the fast-adapting neuron contains more cholinesterase than that of the slow-adapting cell. Neuromuscular endings of some of the accessory and motor fibers are tentatively identified.
CORRELATION OF HISTOCHEMICAL, AUTORADIOGRAPHIC, AND MICRORADIOGRAPHIC DEMONSTRATIONS OF TISSUE CALCIFICATION
Authors: JOHNSTON MURIEL E. , DURBIN PATRICIA W. , ASLING C. WILLET
Published: April 2011

1. The sensitivity and reliability of three representative histochemical tests for calcium (von Kóssa, ferrocyanide, and alizarin) have been compared by applying them to vertebral ossification centers in the rat fetus.

2. The histochemical results were controlled by concurrent study of calcium45 autoradiographs and of soft x-ray microradiographs of the same or adjacent sections.

3. Microradiographs and von Kóssa-positive reactions produced essentially identical representations of mineralized bone and cartilage.

4. The von Kóssa reaction coincided with high grain density regions of calcium45 deposition in stripping-film autoradiographs but did not reflect the sparser densities.

5. The ferrocyanide reaction showed gradients of staining density corresponding in many sites with the varying degrees of radiocalcium density. Faint ferrocyanide reactions extended beyond the limits of microradiographic mineral opacity.

6. The resolution obtained with contact autoradiographs and alizarin dye-lake histological preparations was inadequate for study at high magnification.
AN AUTORADIOGRAPHIC STUDY OF THE DISTRIBUTION OF ZINC-65 IN RAT TISSUES
Authors: MILLAR M. J. , VINCENT N. R. , MAWSON C. A.
Published: April 2011

A new method of preparing rats for 65Zn autoradiography has been described. By ensuring a low excretion rate of 65Zn, autoradiographs with good cellular detail were obtained. The method used for mounting stripping film prevented contact of water with the tissue except during floating-out of the sections.

Autoradiographs of the lateral lobes of the dorsolateral prostate, epididymis, testis, ileum, pancreas and coat hair follicles are illustrated. 65Zn was concentrated at the base and lumen surfaces of the acinar cells in the lateral lobes of the dorsolateral prostate, in the tail pieces of testicular spermatozoa, in epididymal spermatozoa, in the Paneth cells of the ileum, in the pancreatic islets of Langerhans and in the shaft of the hair follicles. 65Zn distribution in liver, submaxillary glands, esophagus, pituitary, adrenals and kidney is also described.
CHLORANILIC ACID AS A HISTOCHEMICAL REAGENT FOR CALCIUM
Authors: EISENSTEIN R. , WERNER M. , PAPAJIANNIS S. , KONETZKI W. , LAING IRIS
Published: April 2011

Chloranilic acid appears to be, for practical purposes, a specific histochemical reagent for the demonstration of calcium, forming colored crystals in the presence of calcium. Despite the disadvantages of only moderate sensitivity and relatively large crystal size, it proved to be quite useful for this histochemical identification of calcium.
A CYTOCHEMICAL ANALYSIS OF THE LACTIC DEHYDROGENASE-DIPHOSPHOPYRIDINE NUCLEOTIDE-DIAPHORASE SYSTEM IN THE EPIDIDYMIS OF THE MOUSE
Authors: ALLEN JOHN M. , SLATER JUDITH J.
Published: April 2011

The properties of diphosphopyridine nucleotide diaphorase and the lactic dehydrogenase-diphosphopyridine nucleotide diaphorase system in the epididymis of the mouse have been investigated by cytochemical and quantitative methods.

In normal mice diphosphopyridine nucleotide diaphorase activity was homogeneously distributed within the cytoplasm of the epithelial cells of the epididymal canal. A gradient of activity existed along the duct, with the lowest levels of activity appearing in the head segments and the highest levels in the body and tail segments.

The lactic dehydrogenase-diphosphopyridine nucleotide diaphorase system differed in its cytological distribution from diphosphopyridine nucleotide diaphorase. In the cells of the body portion of the epididymal canal this system showed high levels of activity in the cell apex with low levels of activity prevailing in the general cytoplasm. In other areas the system showed low levels of activity homogeneously distributed throughout the cytoplasm.

Cytochemical studies indicated that the activity of diphosphopyridine nucleotide diaphorase was depressed by orchidectomy and restored by testosterone propionate administration. The activity of this enzyme was unaffected by section of the vasa efferentia (vasectomy). The activity of the lactic dehydrogenase-diphosphopyridine nucleotide diaphorase system was depressed by vasectomy and orchidectomy. Testosterone propionate restored activity of tissues from castrated animals to levels prevailing in vasectomized animals but did not re-establish the distribution pattern observed in normal animals.

Quantitative determinations confirmed and extended cytochemical observations.
ACROLEIN-SCHIFF, A NEW STAINING METHOD FOR PROTEINS
Authors: VAN DUIJN P.
Published: April 2011

A cytochemical method based on the reaction of acrolein with tissue compounds followed by staining with the Schiff reagent is described. The reaction is completely blocked by acetylation.

Filter paper spot tests of carbohydrates, nucleic acids and proteins revealed a high specificity for proteins. All proteins except the argininerich protamines were positive in the concentrations tested.

Carbohydrates, as well as ribonucleic acid, were negative; deoxyribonucleic acid reacted weakly, probably as a result of a contamination. Tests with compounds of low molecular weight revealed that amino acids were positive, nucleic acid derivatives negative. Arguments are given that the double bond of acrolein reacts with SH, with aliphatic NH2 and NH groups, and with the imidazole group.

The use of this method as a cytochemical stain in combination with Feulgen, thionine-SO2 staining is indicated, especially for chromosome studies. The method also seems applicable to paper and agar electrophoresis.
THE SECTION FREEZE-SUBSTITUTION TECHNIQUE: I. METHOD
Authors: CHANG JEFFREY P. , HORI SAMUEL H.
Published: April 2011

A new technique of freeze-substitution, called "Section Freeze-Substitution," is described. Thin pieces of tissue are quick-frozen as for freeze-drying, and sectioned in a cryostat. The free sections are kept in dry ice-temperature absolute acetone overnight, then picked up with cover glasses, coated with celloidin if necessary, and air dried. The sections adherent to the cover glasses can be stained or incubated in substrate solution directly at room temperature (23-27°C).

Finished slides can be made front fresh tissues

[See figure in the PDF file]

within 24 hours. With few exceptions, both oxidative and hydrolytic enzymes, soluble isotopes, and other chemical substances can be precisely demonstrated in a single substitution process. The cytology of the frozen-substituted tissue is excellent. No other histochemical tissue processing method preserves so much of a particular enzyme. The procedure is simple and the results are highly reproducible.
HISTOLOGICAL STUDY OF DISTRIBUTION OF FLUORESCENT SERUM PROTEINS IN CONNECTIVE TISSUE
Authors: MANCINI R. E. , VILAR O. , GOMEZ C. , DELLACHA J. M. , DAVIDSON O. W. , CASTRO A.
Published: April 2011

Rat serum proteins were labelled in vitro with a fluorescent dye, acid rhodamine B, C.I. No. 45100, using Chadwick's method. Labeled serum was injected intravenously in albino rats weighing 150-200 gm, in doses of 0.8 cc/100 gm body weight. Biochemical and histological findings indicate that there is an equilibrium among serum proteins, connective tissue (as temporary place for storage of these) and reticuloendothelial system, thyroid, ovary and renal tubuli contorti cells (as a place for prolonged deposit of serum proteins).
INTERACTION OF ACIDIC AND BASIC DYES AND BEEF CORNEAL STROMA
Authors: KERN HAROLD L. , GRANT W. MORTON
Published: April 2011

Acidic and basic dyes react preferentially with the mucoproteins of beef corneal stroma. Free carboxyl groups of the mucoproteins can combine readily with cations; additional reaction with ions seems to involve an unmasking of charged groups in these proteins. Most of the dyes studied apparently react as aggregates in 0.02 M solution, and are fully dissociated only in much more dilute solution. The state of aggregation of methylene blue has a pronounced effect on the affinity with which this dye combines with the tissue, but the chemical reaction is limited, nevertheless, to the corneal mucoproteins.
ROLE OF ESTERATIC INHIBITION ON LOCALIZATION OF ESTERASE AND THE SIMULTANEOUS CYTOCHEMICAL DEMONSTRATION OF INHIBITOR SENSITIVE AND RESISTANT ENZYME SPECIES
Authors: SHNITKA THEODOR K. , SELIGMAN ARNOLD M.
Published: April 2011

Evidence is presented to show that the selective demonstration of perinuclear and peribiliary cytoplasmic droplets in rat kidney and liver by Holt with halogen-substituted indoxyl acetates is largely due to inhibition of a sensitive esterase in the cytoplasm in excess of the inhibition of a resistant esterase in the droplets. The inhibition comes from the incorporation of ferro-ferricyanide in the substrate medium to serve as an oxidation catalyst for the capture reaction. Analogous results were obtained by using other substrates such as α-naphthyl acetate and naphthol-AS acetate and other inhibitors such as formalin fixation, di-isopropyl fluorophosphate, NaF and arsanilate.

A product of this study was the development of a method for the simultaneous cytochemical demonstration of inhibitor-sensitive and inhibitor-resistant species of esterase, by taking advantage of red and blue azo dyes that can be formed from naphthol-AS. The distribution of these two species of esterase is described in some detail in rat kidney and liver and the findings are discussed in relation to biochemical data from cell fractionation studies.
OBSERVATIONS CONCERNING THE PRESENCE OF ISOCITRIC DEHYDROGENASE IN MITOCHONDRIA
Authors: SHEPHERD JAMES A.
Published: April 2011

The intracellular distribution of rat and human liver isocitric dehydrogenase is essentially the same as that of mouse and rabbit liver. This enzyme is largely cytoplasmic but washing does not remove all of the activity from the mitochondria.

A method is outlined using permeability considerations as a test of whether an enzyme is within the mitochondria or adsorbed thereon. This method, when applied to washed liver mitochondria, indicates that some isocitric dehydrogenase activity is within the liver mitochondria of rat and rabbit. The results were not conclusive with human liver.
ANALYSIS OF REACTION OF PHOSPHORYL CHLORIDE WITH TISSUE COMPONENTS
Authors: HAUST M. DARIA , LANDING BENJAMIN H.
Published: April 2011

Variation of the ratio of pyridine to neutral solvent (tetrahydrofurane, isooctane or xylene) was employed to investigate the reaction of phosphoryl chloride (POCl3) with tissue components. Two types of products appear to be formed, one (phosphoric acids and (?) amides) reacting with toluidine blue, and another combining with leukofuchsin. Formation of the latter is in general enhanced by the presence of base (pyridine) in the phosphorylation mixture, while that of the former is not. In vitro, compounds with both the =POCl and ≡PO groups react with leukofuchsin in the presence of pyridine, suggesting that a substrate-phosphate-pyridine complex is in part responsible for the direct Schiff reaction after phosphorylation. Despite the wide distribution of reactive substrates, under appropriate conditions selective staining of certain tissue components, e.g., cardiac "colloid," can be obtained.