bluuebunny/crossref_metadata_2025 huggingface dataset:
SOME OBSERVATIONS ON THE HISTOLOGY AND HISTOCHEMISTRY OF THE CHROMAFFIN CELLS PROBABLY STORING DOPAMINE
Authors: FALCK BENGT , HILLARP NILS-ÅKE , TORP ALF
Published: April 2011

The presence of a new type of chromaffin cell in certain tissues in ruminants was reported in previous studies. The results obtained by indirect methods indicated that the cells store dopamine and that this amine is responsible for their chromaffinity. The concentrations of adrenaline, noradrenaline, 3,4-dihydroxyphenylalanine and 5-hydroxytryptamine were found to be too low to account for the chromaffin reaction. This paper is concerned with the general characteristics and the histochemical behaviour of the cells (for simplicity the cells are called dopamine cells).

The dopamine cells contain cytoplasmic granules which may be selectively stained by a nuclear fast red staining method. By use of this method they are clearly distinguishable from other mammalian chromaffin cells.

In all essential respects the dopamine cells resemble the catechol amine containing cells in the adrenal medulla. Their cytoplasmic granules show a direct chromaffin reaction with bichromate but no reaction after pretreatment with formalin. The formalin-fixed cells are argentaffin and strongly reduce ferricyanide, and retain their reducing capacity at least partly even after pretreatment with bichromate. The azo-coupling reaction with diazo-safranin is negative. The dopamine cells are clearly distinguished from the enterochromaffin cells by the chromaffin and azo-coupling reactions.

The most significant result is that the dopamine and noradrenaline cells, after fixation in formalin or bichromate, have the same histochemical behavior. Since dopamine is chemically closely related to noradrenaline, this is what should be expected if the specific chromaffin cells in ruminant tissues contain dopamine.

It is concluded that the results provide further support for the view that the specific chromaffin cells in ruminants store dopamine.
TETRAZOLIUM STUDIES ON THE RETINA: I. INTRODUCTION AND TECHNIQUE
Authors: KUWABARA TOICHIRO , COGAN DAVID G.
Published: April 2011

The retina has physical dimensions that make it especially favorable for incubation studies in its unsectioned state.

With blue tetrazolium anti suitable substrates, dehydrogenase activity is demonstrated in various, but characteristic, forms and places. A red color that depends on enzymatic activity is found in the reticular layers, nerve fiber layer, and lipid portions of the retina. This appears to represent a solubilization of reduced and partially reduced formazan in the lipid portions of the retina. The blue precipitate is found in mitochondria-like granules within certain cells, notably in the ellipsoids of the rods and cones, glial cells, Müller's cells, and to some extent in ganglion cells. It is also found as amorphous and rod-like crystalline deposits in the inner layers of the retina and as a fine dust-like deposit in the nerve fiber layer.

Metabolic activity in the retina as reflected by tetrazolium precipitation persists for hours after death and is present about equally in peripheral and central regions of the retina. The reduction brought about by the utilization of specific substrates representing intermediates in the major metabolic pathways will be the subject of subsequent papers of this series.
ENZYMES CATALYZING SEQUENTIAL REACTIONS IN MOUSE BRAIN AND LIVER SUPERNATANT FRACTIONS: II. THE USE OF PHENAZINE METHOSULFATE
Authors: SHOWACRE JANE , DU BUY HERMAN G.
Published: April 2011

1 . Spectrophotometric and manometric studies of pertinent non-enzymic solutions showed that reduced diphosphopyridine nucleotide is rapidly oxidized by phenazine methosulfate. The reduced phenazine methosulfate (PMS) is gradually oxidized by molecular oxygen. H2O2 is formed. A portion of this H2O2 is spontaneously split into H2O and O2

2. In mouse brain and liver supernatants, with PMS as dye and reduced diphosphopyridine nucleotide (DPNH) as substrate, the same reactions occur. In addition, the formed H2O2 is split by catalase demonstrated to be present in the supernatants used. Th oxygen taken up was a direct measure of the DPNH oxidized.

3. Pyruvate was the main competing electron acceptor studied. When it was added to, or formed by the supernatants, the oxygen uptake during DPNH-oxidation through PMS was reduced, depending on the relative concentration of the electron transferring systems present. In that case the amount of oxygen consumed, when added to the amount of lactate accumulated, was a direct measure of the DPNH oxidized.

4. When oxidized diphosphopyridine nucleotide (DPN) and an appropriate substrate were added to supernatants, the reoxidation by PMS of the DPNH formed by the DPN-requiring dehydrogenase components of a given enzyme group was measured. The oxygen uptake was a measure of the amount of DPNH formed, and thus a measure of the enzymic breakdown of the substrate by the enzyme system under study.

5. When the metabolism of a substrate by the enzyme system under study included the formation of pyruvate, the PMS-dependent oxygen uptake, added to the lactate accumulated, was a measure of the activity of the enzyme system.

6. Homogenized brain tissue yielded a supernatant with fragmented enzyme groups lacking functional flavoprotein. By contrast, as shown previously, this tissue yielded a mitochondrial fraction which retained all of the enzynes necessary for an integrated glucose-metabolizing enzyme system.

7. Homogenized liver tissue yielded a supernatant with flavoprotein linked enzyme systems less fragmented than those found in brain supernatants. At the same time it yields a mitochondrial fraction with an incomplete glycolytic enzyme system.

8. The data support the thesis proposed previously, that the enzymes found in the supernatant fraction of brain and liver cells represent mainly enzymes washed from the mitochondria, and therefore complementary to those retained by these organelles.
A PHOTOMETER HEAD ASSEMBLY FOR USE IN MICROPHOTOMETRY
Authors: BIRGE WESLEY J.
Published: April 2011

This study concerns a new photometer head assembly which has been designed especially for use in visible microphotometry. This assembly consists of three principal units: (1) a Farrand type B photocell mount; (2) a viewing unit modified from the prism housing and side tube of a Leitz Micro-Ibso attachment; and (3) a base plate taken from a Leitz sliding photometer mount. Aside from providing optimum stability and operational convenience, this head assembly meets all of the essential optical requirements necessary for microphotometric work in the visible spectrum. Also, the assemblage of this photometer head piece is greatly simplified since the main component parts utilized in its construction are obtainable from commercial sources.
DISTRIBUTION OF PHOSPHORYLASE IN THE UTERI OF CYCLIC AND HORMONE TREATED RATS
Authors: BO WALTER J.
Published: April 2011

Following the technic of Takeuchi and Kuriaki, phosphorylase activity was identified in the uterus of cyclic and hormone treated rats. In the proestrous uterus phosphorylase activity was more pronounced in the outer longitudinal muscle layer than in the inner circular layer. In the diestrous as well as in the castrate uterus, enzymatic activity was observed in the outer longitudinal muscle layer, and only scattered amounts in the inner circular layer. Following estrogen treatment phosphoryhase activity was very pronounced in both layers, particularly in the outer longitudinal layer. In castrate progesterone-treated rats enzymatic activity was observed in the outer longitudinal layer. In rats that received estrogen and progesterone concurrently, phosphorylase activity was heavy in the outer longitudinal muscle layer and weak in the inner circular muscle layer.

In general, phosphorylase activity in the uterus of the rat corresponds to the distribution of glycogen in the rat uterus. However, enzymatic activity was observed in the uterus when glycogen was not detected by using the periodic acid-Schiff technic.

The distribution of glycogen in the rat uterus is discussed.
A CORRELATIVE STUDY OF THE HISTOCHEMICAL PROPERTIES OF RODENT ACID MUCOPOLYSACCHARIDES
Authors: SPICER S. S.
Published: April 2011

The staining properties of several rodent mucopolysaccharides with alcian blue and with azure A at controlled pH have been surveyed and compared with available radioautographic data of S35O4 incorporation in mucins. The effects of treatment with acid methanol and of sulfation on the staining have been determined. One sulfated, metachromatic and strongly acid type of mucin stains weakly if at all with alcian blue, remaining unstained by alcian blue in the combined alcian blue periodic acid Schiff or alcian blue-azure A (or safranin) procedures, but loses azurophilia while gaining alcian blue reactivity after mild (4 hr 37°) treatment with acid methanol. Some (probably) sulfated mucins stain fully with both dyes and others, presumably also sulfated, color only partially with azure A at pH 1.5, but maximally with alcian blue. The latter two types of substances partially or completely resist supression of either staining reaction by the "mild methylation".

A fourth category stains meta- or orthochromatically at pH 2.5 to 3, or not at all, with azure A, and colors strongly with alcian blue. Some of the latter mucins are sulfated and remain unaltered after mild treatment with acid methanol, whereas others appear to be nonsulfated and lose basophilia reversibly with such treatment. It is suggested that alcian blue staining and metachromasia or orthochromasia with azure A above pH 2.5 which is blocked after 4 hours in acid methanol at 37°C and restored by brief saponification, may be attributed to nonsulfated mucopolysaccharides containing strongly acidic carboxyls. A four hour acidic methylation at 69° eliminates nearly all the basophilia of acid mucins, and saponification restores alcian blue staining to some, but not azurophilia below pH 3.0.

Sulfation of histologic sections renders many neutral and all acid mucins fully chromotropic toward azure A at pH 1.5, but obstructs their alcian blue staining. Exposure to acid methanol after sulfation restores reactivity toward alcian blue and eliminates most artificial azurophilia; but the neutral mucins in Brunner's glands and mucous neck cells of rats and mice remain metachromatic and develop alcian blue affinity. Strong treatment with acid methanol increases the periodic acid Schiff staining of several subfated mucopolysaccharides of the rabbit. Staining with azure A or safranin after alcian blue differentiates alcian blue-negative and -positive sulfated polysaccharides.

Failure of sulfate-free acid mucins to stain with alcian blue above the pH of their dissociation and resistance of the dye complex with these mucins to dissolution by acid or alkali is interpreted as evidence against the alcian blue-substrate complex involving a salt linkage.
THE RELATION OF MITOCHONDRIAL MORPHOLOGY TO SUCCINOXIDASE ACTIVITY AS OBSERVED IN THE RAT KIDNEY AFTER PROTEIN INJECTION
Authors: WALLACE BARBARA JEFFRIES
Published: April 2011

The intraperitoneal injection of native hen's egg white into rats produces morphological changes in the proximal convolutions of the nephron. Normally rod shaped mitochondria are replaced by large dense granules. Previous studies are in disagreement concerning the relation of these granules to the mitochondria. In this work succinoxidase activity was measured using a quantitative tetrazolium method on acetone fixed sections of normal and treated rat kidneys. Osmium fixed sections from the same animals were studied with the electron microscope. At 24 hours after injection intracellular granules in proximal tubule cells are numerous and there is a 35% decrease in succinoxidase activity. The enzymatic decrease is shown to occur only in the proximal conrolutions. With the electron microscope structures are seen which appear to be transitional between mitochondria and granules. At 72 hours after injection the mitochondrial morphology and the enzyme content have returned to normal.
THE HISTOCHEMICAL DEMONSTRATION OF TRACE METALS IN LEUKOCYTES
Authors: MCNARY WILLIAM F.
Published: April 2011

Histochemical methods designed to demonstrate Zn++, Cu++, Co++, Mg++, Mn++, Fe++ and Fe+++ have been applied to blood smears. Metallic elements have been found only in the leukocytes belonging to the myeloid series of cells. The neutrophil was found to contain Zn++ and small amounts of Cu++; the eosinophil, on the other hand, was found to contain large amounts of Cu++ and smaller amounts of Zn++. Basophils were not reported as they could not be identified. Mg++ and Mn++ were present in both the neutrophil and the cosinophil while Co++ was found only in the cosinophil. Iron in either the ferric or ferrous state was not demonstrated in any of the leukocytes in this investigation.
THE LOCALIZATION OF RIBONUCLEASE ACTIVITY IN TISSUE SECTIONS
Authors: DAOUST R. , AMANO HARUKO
Published: April 2011

The substrate film method was applied to localize ribonuclease activity in tissue sections and the distribution of the enzyme in the small intestine, large intestine, pancreas, thyroid, liver and skin of the rat was described.

The resolution of the substrate film method has been improved in the present work by using a technique of film preparation which permitted us to obtain thinner films of substrate.
THE HISTOCHEMISTRY OF SIALIC ACID CONTAINING MUCOPROTEINS
Authors: SPICER S. S. , WARREN L.
Published: April 2011

Sialidase digestion of histological sections releases sialic acid from some sialic acid rich nonsulfated mucins. Such treatment eliminates the metachromatic staining and alcian blue affinity of these mucopolysaccharides. The procedure furnishes a method for histochemical localization of certain sialomucins.
HISTOCHEMICAL DEMONSTRATION OF ACID TRIMETAPHOSPHATASE AND TETRAMETAPHOSPHATASE
Authors: BERG GEORGE G.
Published: April 2011

Trimetaphosphatase is an acid inorganic polyphosphatase whose distribution and properties differ from those of acid and alkaline organic phosphatases. It is the enzyme responsible for previously reported "acid" polyphosphatase" staining. The optimal staining solution for trimetaphosphtase by the Gomori method is: acetic acid 4.5 x 10–2 M, lead acetate 4.6 x 10–3 M, sodium trimetaphosphate 6 x 10–4, M, adjusted to pH 4.8 with nitric acid. Best preservation of enzyme for staining is by post-fixation in the ribbon (dehydrate in cold acetone, embed in paraffin, section without wetting, spread ribbon on 0.02 M magnesium acetate pH 5.9, mount, drain and dry at 40-45°C). Brief standard fixation in cold neutral formaldehyde can also be used. Alcohol inactivates the enzyme. The report compares direct fixation to fixation in the anhydrous block, in the paraffin ribbon, and in the frozen section, and presents the effects of fifteen fixatives. Artifact staining in the Gomori method is shown to be accentuated or suppressed by choice of fixative.
STUDIES ON THE HISTOCHEMICAL DIFFERENTIATION OF ENZYMES HYDROLYZING ADENOSINE TRIPHOSPHATE
Authors: FREIMAN DAVID G. , KAPLAN NANCY
Published: April 2011

At least 3 enzymes or groups of enzymes present in dog kidney and jejunum are capable of splitting adenosine triphosphate at pH 9.4. In addition to nonspecific alkaline phosphatase and what appears to be a true adenosine triphosphatase, an enzyme behaving like a less specific organic polyphosphatase is present in small blood vessels and smooth muscle. These enzymes can be readily differentiated by various inhibitors such as cysteine, ethylenediamine tetraacetic acid, 80% ethanol, pretreatment in acetate buffer at pH 4.0 with or without added chloride or nitrate ion, or by incubation at 4°C. Differential effects can also be obtained by pretreatment with lead or zinc ion at pH 6.0.

Of the substrates tested only inosine triphosphate gives results comparable with adenosine triphosphate. All other substrates, including various nucleotides and thiamine pyrophosphate, produce patterns similar to that obtained with adenosine diphosphate, or with adenosine triphosphate after pretreatment with buffer at pH 4.0. These observations suggest that an enzyme less specific than adenosine triphosphatase is being demonstrated. The similar patterns obtained following incubation in lead-adenosine triphosphate substrate at pH 7.2 also indicate that true adenosine triphosphatase cannot be shown by this method. 5'-Nucleotidase plays little role in these reactions under the experimental conditions used.

Enzymes behaving in similar fashion have been noted in some human tissues. Of particular interest is a broad inner zone of the circular muscle coat in the small intestine of the dog which is strongly positive for adenosine triphosphatase and polyphosphatase, but negative for 5'-nucleotidase. A similar, but much narrower zone has been noted in the ileum and colon of man.
LIPID DISTRIBUTION IN THE TISSUES OF HIBERNATING AND NON-HIBERNATING TOAD (BUFO MELANOSTICTUS)—A HISTOCHEMICAL STUDY I. SUDANOPHILIA, UNSATURATED LIPID AND PLASMALOGEN
Authors: MUKHERJI MRITYUNJOY , DEB CHANDICHARAN
Published: April 2011

Neutral lipids (Sudan positive substances), unsaturated lipids and plasmalogen were demonstrated in liver, fat body, kidney, adrenals, testis, intestine and cardiac muscle of toad ( Bufo melanostictus) during hibernation (February), post-hibernation (April), breeding season (August) and prehibernation (November).

In liver and fat body maximum sudanophilia has been observed during November, with decrease during February and complete disappearance during April. In August it began to accumulate again. Unsaturated lipid and plasmalogen were absent in February and rose to a maximum during August. These changes indicate that fat is being utilized during hibernation. In testis a maximum of unsaturated lipid and plasmalogen have been observed during August and a minimum during February. In February accumulation of neutral fat has been observed in the intertubular and intratubular space of this organ. In adrenals maximum sudanophilia, unsaturated lipid and plasmalogen have been observed during February, with minimums during August. These changes in testes and adrenals indicate hypofunction of the glands during hibernation. In kidney and intestine a maximum of unsaturated lipid and plasmalogen has been seen during August, with a minimum during February; cardiac muscle showed very little alteration in the lipid fractions. These changes have been ascribed to seasonal alteration in the activity of the organs; the heart, being a vital one, showed minimum alteration.
CHROMATOGRAPHIC SEPARATION AND ISOLATION OF METACHROMATIC THIAZINE DYES
Authors: TAYLOR KENNETH B.
Published: April 2011

1. The chromatographic behaviour of 22 closely related N-alkyl thionins in a number of solvent systems is described.

2. Details are given of the separation of commercial mixtures of the azures into their components and the application of this to the analysis of Romanowsky stains.

3. A table giving the Rf equivalents and absorption maxima of the dyes is appended and the additive effect of alkyl substitution is discussed.
THE HISTOCHEMICAL DEMONSTRATION OF ENZYMATIC ACTIVITY BY A NONENZYMATIC REDOX REACTION. REDUCTION OF TETRAZOLIUM SALTS BY INDOLYL-3-ACETALDEHYDE
Authors: GLENNER GEORGE G. , WEISSBACH HERBERT , REDFIELD BETTY G.
Published: April 2011

The availability of synthetic indolyl-3-acetaldehyde has made possible the determination of the mechanism of reduction of tetrazolium salts in the tryptamine-tetrazolium technique for the demonstration of monoamine oxidase activity. This aldehyde, formed as the result of the enzymatically catalyzed oxidative deamination of tryptamine, directly reduces sensitive tetrazolium salts to localize monoamine oxidase activity in tissue sections. No implication of a diaphorase-like system in the reduction of the tetrazoles was found.

The possibility of a pigment precursor being formed as the result of the oxidation of the indolyl aldehyde during tetrazole reduction is discussed and the utilization of a similar scheme of nonenzymatic reduction of an indicator dye for demonstrating both oxidative and hydroytic enzyme systems is presented.
EFFECTS OF COENZYME Q10 AND MENADIONE ON SUCCINIC DEHYDROGENASE ACTIVITY AS MEASURED BY TETRAZOLIUM SALT REDUCTION
Authors: WATTENBERG LEE W. , LEONG J. LIONEL
Published: April 2011

Coenzyme Q10 and menadione have been shown to enhance the succinic dehydrogenase activity of rat liver homogenates and frozen sections as measured quantitatively by the reduction of INT. Increased formazan deposition as estimated by visual examination was also found in sections of liver and other tissues in comparable experiments with the two quinones in which the tetrazolium salt Nitro-BT was employed.

Extraction of sections with acetone decreased the succinic dehydrogenase activity of liver sections by approximately two-thirds of that present in nonextracted sections as measured by INT reduction. The addition of coenzyme Q10 or menadione restored the activity to essentially that present in nonextracted sections incubated in the presence of the same constituents. These data suggest that a quinone, presumably coenzyme Q10, acts as an intermediate electron transport agent between succinic dehydrogenase and INT or Nitro-BT.

The effects of coenzyme Q10 and menadione on the activities of diphosphopyridine nucleotide (DPNH) diaphorase, triphosphopyridine nucleotide (TPNH) diaphorase, α-glycerophosphate dehydrogenase and monoamine oxidase, all of which are demonstrable by tetrazolium salt reduction procedures, have been studied. Two of these enzymes, TPNH diaphorase and monoamine oxidase, show no significant enhancement of activity in the presence of coenzyme Q10, and a relatively slight enhancement in the presence of menadione. α-Glycerophosphate dehydrogenase shows a pattern of enhancement comparable to that of succinic dehydrogenase. DPNH diaphorase activity, while enhanced by both of the quinones, shows a lesser degree than is found with the succinic dehydrogenase and α-glycerophosphate dehydrogenase systems.

The diverse effects of coenzyme Q10 and menadione on the five enzyme systems investigated in the present work indicate that the two quinones do not simply act as nonspecific intermediate electron carriers between dehydrogenase system and tetrazolium salt, but that they enhance enzyme activity by a more specific interaction with some component of the enzyme system.
HISTOCHEMICAL IMPLICATIONS OF ELECTRON MICROSCOPY OF THE CENTRAL NERVOUS SYSTEM
Authors: LUSE SARAH A.
Published: April 2011

The impact of electron microscopy on our concepts of neurophysiology and neurochemistry already has been far reaching. In particular has this been true in the areas of extracellular space, water transport, blood-brain barrier, neurosecretion and synaptic transmission. Its contributions in the field of neoplasia are so recent and so scant that one can only conjecture that the correlation of abnormal structure with abnormal function can be carried over into the cytochemical analysis of the lesions; and that electron microscopy will offer a technic for studying the site of action of the cytotoxic agents.
QUANTITATIVE HISTOCHEMISTRY OF THE CEREBRAL CORTEX
Authors: POPE ALFRED
Published: April 2011

The intralaminar distributions of representative brain enzymes have been determined in rat somatosensory and human frontal isocortex by means of quantitative microchemical techniques.

Results of experiments upon cytochrome oxidase, l-alanylglycine dipeptidase, acetylcholinesterase, and two adenosinetriphosphatases in rat cortex are described and their anatomical significance discussed.

The essentially similar intracortical patterns of the same five enzymes in human frontal isocortex suggest uniformity in the architectonic organization of metabolic events in mammalian cerebral cortex.

The value and limitations of microchemical analyses upon microgram-sized tissue samples are briefly discussed, especially with respect to their usefulness in neurochemistry.
ACTIVITY OF RIBONUCLEASE IN PREPARATIONS OF TESTICULAR AND STREPTOCOCCAL HYALURONIDASE
Authors: LOVE ROBERT , LILES ROSCOE H. , ELLEM KAY A. O.
Published: April 2011

1. Ribonuclease activity has been demonstrated biochemically or histochemically in a number of preparations of bovine testicular and streptococcal hyaluronidase.

2. A number of properties of the hyaluronidase-ribonuclease have been demonstrated and compared with those of pancreatic ribonuclease and hyaluronidase.

3. The activity of the hyaluronidase-ribonuclease in aqueous solutions of testicular hyaluronidase was selectively inhibited by oxidation with hydrogen peroxide or by the addition of small amounts of metallic cations.

4. The activity of hyaluronidase was destroyed by heat without affecting that of hyaluronidase-ribonuclease.

5. The intensity of staining of hyaluronic acid in 1% casein and of the acid mucopolysaccharides of cock's comb by toluidine blue was increased by treatment with ammonium molybdate.
THE CHEMICAL SPECIFICITY OF THE SCHULTZ TEST FOR STEROIDS
Authors: LEWIS P. R. , LOBBAN MARY C.
Published: April 2011

The colours given by a number of pure steroids under various strongly acid conditions have been studied both in the test tube and in model sections prepared in various ways. Aqueous sulphuric acid solutions containing ferric ions were found to give an intense blue-green colour with steroids closely related chemically to testosterone, and colours in the pink-mauve range with certain other steroids such as the oestrogens and pregnenolone. The blue-green colour has two sharp absorption bands in the red which make possible the conclusive identification of the testosterone group of compounds. A new modification of the Schultz reaction has therefore been devised: frozen sections of formalin-fixed material are mounted on slides, blotted dry, treated with a drop of 80% aqueous sulphuric acid containing 0.5% iron alum and the resulting colours observed. This procedure has several advantages over previous modifications: there is no charring and very little cytological distortion; the colours are stable for many hours, and their chemical specificity is more clearly defined. Cholesterol itself gives no colour, but preliminary oxidation (e.g. by preincubation in iron alum) leads to the formation of the typical blue-green colour. Various aspects of this new histochemical modification of the Schultz reaction are discussed.
GROWTH, FLUORESCENCE AND METACHROMASY OF CELLS CULTURED IN THE PRESENCE OF ACRIDINE ORANGE
Authors: WOLF MERRILL K. , ARONSON SAMUEL B.
Published: April 2011

Four types of animal cells were cultured in perfusion chambers in medium containing acridine orange and serially observed by phase and fluorescence microscopy, so that cellular injury from toxic and photodynamic effects of acridine orange could be correlated with the variable fluorescence images of the cells. Spontaneous movements of pigment granules in healthy chorioidal and ciliary melanocytes provided a valuable criterion of cellular health. Dye concentrations of 10 –6 gm/ml permitted healthy outgrowth of brilliantly fluorescent cells for at least 20 days, but higher concentrations were lethal. Uninjured living cells had orthochromatic (green) fluorescence. Reversibly injured cells acquired metachromatic (red-fluorescent) cytoplasmic granules which might be identical with mitochondria or with the neutral red granules of Jackson. With irreversible photodynamic injury, ribonucleic acid of nucleoli and cytoplasm stained metachromatically; deoxyribonucleic acid of nuclei alnsost always remained orthochromatic. As degeneration ensued, cells lost first their metachromasy, finally all their fluorescence. The variable fluorescence images of living, injured, killed, degenerated and fixed cells stained with acridine orange may be explained according to the theory of variable stacking of bound dye molecules.
CYTOLOGICAL LOCALIZATION OF GLYCOGEN IN CULTURED SKELETAL MUSCLE
Authors: ENGEL W. KING
Published: April 2011

The periodic acid Schiff stain was used to demonstrate the cellular location of glycogen formed and stored by chick embryo skeletal muscle cells newly grown in vitro without innervation. No glycogen was found in single myoblasts. In the myoblastic and less mature myocytic straps, there were irregular-sized granules of glycogen in the cytoplasm, especially in the perinuclear areas. Much glycogen was formed in the more mature cultured myocytic straps, and in a pattern like that of innervated adult skeletal muscle cells in vivo. There was glycogen in the perinuclear and subsarcolemmal cytoplasm and in alternating wide and narrow bands, tentatively interpreted as I and M bands respectively.
METAL CHELATE REACTION OF ENTEROCHROMAFFIN
Authors: LILLIE R. D.
Published: April 2011

Enterochromaffin slowly binds ferrous ions at pH levels between 3.5 and 5.0 and gives them up again at low pH levels (1-2.5). Ferrous ions so bound may be demonstrated in situ by action of potassium ferricyanide, probably by acid dissociation of the chelate complex and precipitation of Turnbull's blue. This reaction is prevented by acetylation and tosylation, in agreement with the azo reaction. It is prevented by drastic oxidations but not by milder, sufficient to prevent azo coupling.

This reaction was at first thought to indicate the presence of a substituent ortho to the phenolic hydroxyl of enterochromaffin, but in view of the occurrence of the reaction in model experiments with some para substituted phenols and phenylamines, after formaldehyde treatment, this conclusion must remain indefinite.

Formaldehyde fixed serotonin model sections reacted relatively weakly, though giving strong reduction, azo and hematoxylin reactions.
CYTOPHOTOMETRIC STUDY ON THE CHROMAFFIN REACTION, THE IODATE REACTION AND FORMALIN-INDUCED FLUORESCENCE AS INDICATORS OF ADRENALINE AND NORADRENALINE CONCENTRATIONS IN THE ADRENAL MEDULLA
Authors: ERÄNKÖ OLAVI , RÄISÄNEN LIISA
Published: April 2011

Variable depletion of adrenal catecholamines was caused in rats by injections of reserpine and muscular work. The intensities of the chromaffin reaction, the iodate reaction and formalin-induced fluorescence in adrenal sections were measured by cytophotometry and compared with chemically determined concentrations of adrenaline and noradrenaline in the adrenal medulla. A close correlation was observed between the optical density of the chromaffin reaction and the total catecholamine concentration, as well as between the intensity of the formalin-induced fluorescence, the optical density of the iodate reaction and the noradrenaline concentration.
INVESTIGATIONS INTO THE HISTOCHEMISTRY OF CHITIN
Authors: RUNHAM N. W.
Published: April 2011

1. Investigations into the histochemistry of chitin are described.

2. Chitin can be identified by its positive periodic acid Schiff, Hale, and alcian blue reactions. The periodic acid Schiff reaction is decreased by deamination, and the alcain blue and Hale reactions are blocked by methylation and acetylation procedures. Methylation increases considerably the very weak bromphenol blue reaction.

3. The Hale and alcian blue reactions of chitin are probably due to the formation of chelate complexes.

4. Methylation of chitin apparently causes deacetylation.

5. Deamination of chitin appears to result in a molecular rearrangement of the glucosamine molecule.

6. The importance of these reactions in the histochemical analysis of polysaccharides is discussed.
PREPARATION OF NAPHTHOL AS-TR PHOSPHATE FOR LOCALIZATION OF PHOSPHATASE
Authors: JEFFREE GRACE M. , TAYLOR KENNETH B.
Published: April 2011

The use of PCl5 in dioxan for the conversion of Naphthol AS-TR to the acid phosphate, even without the addition of catalysts other than pyridine, gave a conversion considerably superior to that obtained using POCl3 in either tetra-hydrofuran or dioxan, with or without the additional catalysts suggested by Burstone. The product proved satisfactory for histochemical localization of alkaline phosphatase in bone, though it is suspected that a certain amount of polymerization had occurred in the process of phosphorylation.
A METHOD FOR THE CYTOPHOTOMETRIC ESTIMATION OF RIBONUCLEIC ACID
Authors: RITTER CARL , DI STEFANO H. S. , FARAH A.
Published: April 2011

A staining procedure is described in which the basic dye crersyl violet is shown to possess properties which makes its use advantageous in the staining and cytophotometric estimation of ribonucleic acid. Cresyl violet is shown to stain ribonucleic acid quite specifically when used at pH 4.2, and the resulting in situ staining is shown to follow the Beer-Lambert Law over the range of thicknesses and concentrations found in microscopic sections.
HISTOCHEMICAL STUDIES ON HUMAN MUSCULAR DYSTROPHY
Authors: GOLARZ M. NELLY , BOURNE GEOFFREY H. , RICHARDSON H. DALE
Published: April 2011

The proliferating connective tissue of muscular dystrophy can be shown by histochemical methods to contain enzymes which hydrolyse many high energy phosphates and phosphorylated co-enzymes, but which do not affect phosphorylated glycolytic intermediates. These results are discussed and the suggestion is made that the possibility of muscular dystrophy being primarily a connective tissue disease should be seriously considered.

This work was carried out under a grant from the Muscular Dystrophy Associations of America Inc. and under a U. S. Public Health Service grant no. B2038. Some of the equipment with which the work was carried out was purchased with funds made available by the Aid to Muscular Dystrophy Research Chapter, Boston, Mass. of the National Foundation for Neuromuscular Diseases.
AN INVESTIGATION OF THE FACTORS INFLUENCING THE METACHROMATIC STAINING OF MAST CELL GRANULES
Authors: RADDEN B. G.
Published: April 2011

1. In the mast cell, the staining of metachromatic granules is influenced by the pH of the solution—at a level of pH 3 metachromatic granules stain rapidly, and orthochromatic staining is retarded.

2. The presence of metallic cations delays granule staining, the duration of the delay being proportional to the cationic concentration, related to the cationic valency, and to the position of the cation in its valency group.

3. Granule staining is unaffected by the addition of metallic cations to the "pretreatment" solution, and accelerated by temperature increase.
STUDIES OF INDOPHENOL BLUE SYNTHESIS: II. THE "G" AND "M" NADI REACTIONS IN THE RAT MAJOR SALIVARY GLANDS
Authors: PERSON PHILIP , SCHNEIDER ROSE MARY , SCAPA SYLVIA
Published: April 2011

1. Indophenol blue synthesis has been studied in fresh frozen sections and formalin-treated fresh frozen sections of the major rat salivary glands.

2. In fresh frozen sections ("G" Nadi reaction), indophenol-blue synthesis was consistently more rapid and more intense in ducts and tubules than in acini.

3. 10% formalin treatment of fresh frozen sections completely abolished indophenol blue synthesis by duct and tubule structures in all glands. However, after approximately one hour of incubation of these treated sections, indophenol blue appeared ("M" Nadi reaction) in the acini of submaxillary and parotid glands, but not in the acini of the sublingual gland.
THE EXPERIMENTAL ERROR OF FEULGEN CYTOPHOTOMETRY IN THE ANALYSIS OF BULL SPERMATOZOA OVER AN EXTENDED PERIOD OF TIME
Authors: WELCH ROBERT M. , HANLY E. W.
Published: April 2011

Feulgen cytophotometry has been applied over a period of about two years to the analysis of the deoxyribonucleic acid content of 658 samples of spermatozoa from 275 Santa Gertrudis bulls of the King Ranch, most of which were under two years of age. A system of correction for smear to smear and slide to slide staining variation due to experimental error is presented that restricts the total maximum experimental error of a single determination to 15%, and to 10% in 80% of samples, when 18-25 sperm are measured per smear. Refinements of technique are described by which the maximum error can be reduced to 7% in 95% of samples and to 5% by increase in the number of sperm measured per sample smear.
A HISTOCHEMICAL AND RADIOAUTOGRAPHIC STUDY OF THE PARTICIPAPATION OF FIBROBLASTS IN THE PRODUCTION OF MUCOPOLYSACCHARIDES IN CONNECTIVE TISSUE
Authors: MANCINI R. E. , VILAR O. , STEIN E. , FIORINI H.
Published: April 2011

A histochemical and radioautographic study of the connective tissue of rat skin was carried out from birth to maturity. The following observations were made:

1) Early fibroblasts changed into typical adult connective tissue cells, fibrocytes, with gradual diminution of cytoplasmic nucleoproteins, periodic acid Schiff positive material, succinodehydrogenase, β-glucuronidase and leucyl-aminopeptidase.

2) Lack of striking morphological or cytochemical changes of the mast cells in the different ages as compared with fibroblasts, but more intense enzymatic reactions with respect to succinodehydrogenase, β-glucuronidase, leucyl-aminopeptidase, alkaline and acid phosphatase, lipase esterase and cytochrome oxydase were found.

3) Using adenine-C14 and methionine-S35, the localization of these substances was seen more in the fibroblasts and mast cells than in the intercellular ground substance; in the fibroblasts the uptake was higher in the embryonic and early postnatal connective tissues than in the adult.

4) The embryonic and early postnatal connective tissues to synthesize sulphated polysaccharides trapped more S35 than the adult. S35 was seen first in the fibroblasts and then in the intercellular ground substance. The same results were observed in tissue cultures of fibroblasts. Mast cells incorporated and stored more S35 than fibroblasts, but there was no appreciable difference in their capacity for this at various ages, as there was in fibroblasts.
NEW DIAZONIUM COMPONENTS AS COUPLING AGENTS IN THE DEMONSTRATION OF PHOSPHATASES
Authors: BURSTONE MARVIN S. , WEISBURGER ELIZABETH K.
Published: April 2011

A histochemical technique for the diazotization of highly insoluble amines is described. The diazotization procedure is carried out in the presence of inert solvents, dimethylformamide or demethylsulfoxide.

Most of the amines contain coordinating groups which facilitate chelation of the final dyestuff with metals, including copper and cobalt. The aforementioned coordinating groups include nitrogen, keto, hydroxyl, amino, carboxyl, and the azo grouping. Several dye chelates were found to be highly resistant to xylene and ethanol.

In addition to the successful application of chelation techniques to azo-dye enzyme histochemistry, the first use of a metal containing diazonium salt is reported. The compound is diazotized p-(acetoxymercuri)-aniline. Diazotized 3-aminofluoranthene is highly stable in solution and is suitable for use in simultaneous coupling techniques.

Use of complex diazotized amines possessing coordinating groups for subsequent chelation enhances chromogenicity and fineness of localization and may find application in electron microscopy.
HISTOCHEMICAL DEMONSTRATION OF URIDINE DIPHOSPHATE GLUCOSE-GLYCOGEN TRANSFERASE IN ANIMAL TISSUES
Authors: TAKEUCHI TADAO , GLENNER GEORGE G.
Published: April 2011

Polysaccharide was histochemically synthesized in muscle fibers and other tissue cells by using uridine diphosphate glucose as substrate. By revising the method previously described in light of new biochemical and histochemical findings more satisfactory optimal conditions were defined for the demonstration of the uridine diphosphate glucose-glycogen transferase enzyme.

The histochemical properties of the reaction were very similar to those reported biochemically and this tended to verify the specificity of the reaction. Glucose-6-phosphate stimulated intensely the uridine diphosphate glucose-glycogen transferase reaction in an alkaline range.
THE HISTOCHEMICAL LOCALIZATION OF ACETYLCHOLINESTERASE IN THE FINE STRUCTURE OF NEUROMUSCULAR JUNCTIONS OF MOUSE AND HUMAN INTERCOSTAL MUSCLE
Authors: ZACKS S. I. , BLUMBERG J. M.
Published: April 2011

The localization of acetylcholinesterase activity in the fine structure of mouse and human neuromuscular junctions was studied by the method of Barrnett and Palade. A formolsucrose solution was used for primary fixation followed by osmification. It was found that deposits of lead sulphide, the product of the histochemical reaction, were found in axonal and sarcoplasmic mitochondria and most prominently in the primary and secondary synaptic clefts of the subneural apparatus.

Rarely were reactive sites found near the M-band. Control experiments utilizing inhibitors and high concentrations of Pb(NO3)2 were performed.

The possible significance of acetylcholinesterase activity in the areas described is discussed.
HISTOCHEMISTRY OF THIOLACETIC ACID ESTERASE: A COMPARISON WITH NONSPECIFIC ESTERASE WITH SPECIAL REGARD TO THE EFFECT OF FIXATIVES AND INHIBITORS ON INTRACELLULAR LOCALIZATION
Authors: WACHSTEIN MAX , MEISEL ELIZABETH , FALCON CARMEN
Published: April 2011

In confirmation of previous reports, the thiolacetic acid technique represents a simple and reliable method for the demonstration of motor endplates in striated muscles. This type of esterase is present in many other locations as well. When its distribution pattern was compared with that for nonspecific esterase, it was found to be identical, although in some locations nonspecific esterase was active in additional sites. The effect of various inhibitors on the staining reaction, which was carried out in most instances in sections fixed in chilled Baker's CaCl2 formalin, was also quite similar. A notable exception, however, was observed in the pancreas of various species. In contrast to the marked activity of nonspecific esterase in the acinar cells, no staining was noticed in sections incubated in the thiolacetic acid medium. However, addition of sodium taurocholate converted this negative reaction into a strong positive one, indicating that under this circumstance a true lipase was visualized. Following the treatment of sections from rat kidney and liver with organophosphorous compounds, particularly E 600 (diethyl- p-nitrophenylphosphate), cytoplasmic activity was suppressed and staining in droplets became very noticeable in sections prepared for the demonstration of both nonspecific and thiolacetic esterase. Thus, these structures at present identified with de Duve's lysosomes contained a different kind of esterase (C esterase of Pearse with a cathepsin-like activity). The implications of these findings are discussed in reference to the significance of the intercellular localization of esterase and the differentiation of various esterases on the basis of organophosphorous inhibitors. It is concluded that thiolacetic acid esterase under histochemical conditions is hydrolyzed by acetylcholinesterase at least in the motor endplates, by B or aliesterase in the cytoplasm, and by type C esterase in the lysosomes and under appropriate circumstances also by pancreatic lipase.
RAPID PREPARATION OF FROZEN SECTIONS FOR COMPARATIVE HISTOCHEMICAL STUDIES: JUMP-MOUNTING OF PAIRED FROZEN SECTIONS ON SEMI-HEATED MICROSLIDES
Authors: IRONS IAN I.
Published: April 2011

A technique for rapidly preparing a large number of paired and uniformly good quality frozen sections has been described in terms of specific procedures followed and apparatus required. Two innovations were noted: 1) a microtome chuck that allows precise alignment of two tissue blocks that are to be cut simultaneously, and 2) a heating tray for microslides that enables a controlled amount of heat to be applied to a small section of a slide that is subsequently used in "jump" mounting of two freshly cut sections.
HISTOCHEMICAL REACTION OF AN AROMATIC DIAMINE WITH ACID GROUPS AND PERIODATE ENGENDERED ALDEHYDES IN MUCOPOLYSACCHARIDES
Authors: SPICER S. S. , JARRELS M. H.
Published: April 2011

Both aqueous and alcoholic variants of a periodic acid N,N-dimethyl- p-phenylenediamine method selectively impart color and fluorescence to neutral mucopolysaccharides. The aqueous method reveals differences in reactivity of various neutral polysaccharides. Prolonged staining of histologic sections with the aqueous variant superimposes a different coloration of acid mucins. This procedure offers advantages in differentiating periodate reactive from periodate negative acid mucopolysaccharides and in demonstrating mixtures of acid and neutral mucins in certain organs and cells.

Removal of basophilia by active methylation or sialidase digestion changes the periodate diamine staining of acid mucins to that characteristic of neutral mucins; sulfation of tissue sections induces the converse effect.
ENZYMORPHOLOGY: A STUDY OF FREE CELLS
Authors: FISHMAN WILLIAM H. , LADUE K. T. , BORGES P. R. F.
Published: April 2011

A technique has been developed for the enzymorphologic study of five enzymes in free cells. The material investigated was an ascitic form of a mouse monocytoid leukemia (C-1498), Ehrlich ascites carcinoma and human blood leukocytes. Preservation of the integrity of cell morphology and of enzyme activity was accomplished by the use of a formaldehyde-chloralhydrate fixative followed by embedding in Knox gelatin. On refrigeration, solid gelatin blocks embedding suspended cells result. These may be mounted on a freezing microtome and 5 µ sections readily cut and handled.

The location of β-glucuronidase, acid phosphatase, alkaline phosphatase, diphosphopyridine nucleotide-diaphorase and α-naphthyl esterase in these various cells has been described in detail. For β-glucuronidase, diphosphopyridine nucleotide-diaphorase and acid phosphatase, consistent findings of cell enzymorphology are reported for the first time.

The technique lends itself to application for all types of cell populations. It also has the great advantage that the same population of cells in gelatin can be subjected to repeated scrutiny over a period of at least three weeks. The conditions of fixation economize on cell material in that a different fixative for each enzyme has become unnecessary, as has also the restriction of working with fresh frozen material prior to performing the reaction for diphosphopyridinenucleotide-diaphorase.
AN IMPROVED SAKAGUCHI REACTION FOR MICROSPECTROPHOTOMETRIC USE
Authors: DEITCH ARLINE D.
Published: April 2011

1. The Sakaguchi reaction for arginine has been modified to provide a technique suitable for the quantitative microspectrophotometric estimation of protein arginine. The reagent consists of 2,4-dichloro-α-naphthol, sodium hypochlorite and barium hydroxide. Previous Sakaguchi reagents containing high concentrations of sodium or potassium hydroxide caused extensive damage to the tissue; substitution of barium hydroxide has eliminated this problem. In addition, it appears that barium ions stabilize the resulting Sakaguchi color.

2. Conditions were found for making intensely stained, stable and reproducible preparations which can be dehydrated and mounted to match their index of refraction without loss of colour, and which do not fade significantly for at least two weeks.

3. Intensive acetylation and formalinization, which are thought to block the guanidyl group of arginine, considerably decrease the intensity of the Sakaguchi reaction.

4. The ratio between the amount of Sakaguchi color in bull sperm and in trout sperm, as determined from the results of microspectrophotometric examination, is in rather close agreement with the ratio derived from the amounts of arginine in these cells estimated biochemcially. This supports the concept that the Sakaguchi reaction described here represents a stoichiometric reaction with arginine.
THE CHROMATOGRAPHIC PURIFICATION OF FLUORESCEIN-ANTIBODY
Authors: CURTAIN C. C.
Published: April 2011

Crude fluorescein antibody-globulin conjugates may be freed of soluble fluorescein derivatives and other low-molecular weight by-products by filtration through a Sephadex column of G25. Nonspecifically staining conjugate may be removed by filtration of the fluorescein-globulin through a column of N,N-diethlaminoethyl cellulose in pH 6.0 0.02 M phosphate buffer. The method may be used for either purifiying fluores and antibody or for isolating the non-specifically staining components of the crude conjugates.