bluuebunny/crossref_metadata_2025 huggingface dataset:
STUDIES ON THE FEULGEN REACTION WITH HISTOCHEMICAL MODEL SYSTEMS
Authors: HARDONK M. J. , VAN DUIJN P.
Published: April 2011

The absorption spectra of Feulgen-stained deoxyribonucleic acid (DNA)-containing membranes have been studied under various conditions. The absorption curve of the Feulgen-stained DNA-cellulose differs from those of the other model systems and is comparable to that of a solution of apurinic acid stained by the Schiff reaction. The spectral shift caused by the reaction of formaldehyde and SO2 with the stained films was used as a criterion for the degree of substitution of the bound pararosaniline. The spectrum of DNA-cellulose, unlike the spectra of the other models, did not show any change under the action of these reagents. This indicates that the three amino groups of pararosaniline are all bound to the DNA in a stained DNA-cellulose film.

When the mobility of the apurinic acid chain is restricted, the formation of the trisubstituted product is apparently hindered. Such a situation may be present in DNA-containing polyacrylamide films containing proteins and also in cell nuclei.

From a study of the literature it is concluded that the shoulder in the pararosaniline spectrum is most probably caused by a configurational isomer of the dye as indicated by Lewis et al. (16) for crystal violet. To explain the abnormal spectrum of the stained DNA-cellulose, it is proposed that in this case pararosaniline is bound with the three amino groups to three neighbouring deoxyribose groups in the hydrolyzed DNA. Studies with Stuart models of the molecules involved showed that in this case one conformation isomer of pararosaniline can be constructed more easily than the other. This situation may explain the spectrum of the stained DNA-cellulose. The other spectral results could also be interpreted on basis of this hypothesis.
ULTRARAPID TISSUE FREEZING IN LIQUID NITROGEN
Authors: MOLINE S. W. , GLENNER G. G.
Published: April 2011

Tissue, powder-coated prior to immersion in liquid nitrogen, cools to a center temperature of –50°C four to five times faster than comparable tissue immersed in ethylene glycol monomethyl ether (Cellosolve®, Union Carbide Corp.) at –79°C and twice as fast as tissue immersed uncoated in isopentane-liquid nitrogen (–155°C) or liquid mitorgen. The morpohlogical preservation of tissue structure when this method is used on blocks of kidney, pancreas, and liver, for either enzymic reactions or oversight staining technieques, is comparable to that obtained with hydrocarbon baths cooled in liquid nitrogen. The coating method is recommended as a substitute, rather than as a replacement, for hydrocarbon baths cooled in liquid nitrogen, when circumstances demand the advantages of safety, simplicity and speed.
A TECHNIQUE FOR LIGHT AND ELECTRON MICROSCOPIC IDENTIFICATION OF ADRENALIN- AND NORADRENALIN-STORING CELLS
Authors: TRAMEZZANI JUAN H. , CHIOCCHIO SARA , WASSERMANN GUILLERMO F.
Published: April 2011

Fixation of adrenal glands with 6.5% glutaraldehyde in Millonig buffer at pH 7.2 for 1 or more hours results in a yellow color in noradrenalin-storing cells while adrenalin-containing cells remains uncolored. When frozen sections of adrenal gland fixed in glutaraldehyde are treated with an ammoniacal silver solution, silver is reduced by noradrenalin cells in less than 30 seconds.

A deep brown or black color according to the silver solution used, appears in noradrenalin-containing cells while the adrenalin ones remain uncolored.

Based on these facts a technique for light and electron microscopic localization of noradrenalin has been developed.

The results permit the following interpretation of the glutaraldehyde silver reaction for noradrenalin-containing cells. The condensation of glutaraldehyde with noradrenalin forms a yellow azomethine. Whereas this compound is insoluble adrenalin will not combine with glutaraldehyde and remains soluble. During washing adrenalin therefore disappears from sections while the yellow glutaraldehyde-noradrenalin compound remains. The aldehyde group of this compound reduces ammoniacal silver solutions.
THE COMBINED USE OF MICROINCINERATION AND THE PRUSSIAN BLUE REACTION FOR A MORE SENSITIVE HISTOCHEMICAL DEMONSTRATION OF IRON
Authors: FENTON R. H. , JOHNSON F. B. , ZIMMERMAN L. E.
Published: April 2011

The Prussian blue reaction was found useful for the demonstration of iron in the celloidinized ash left after direct microincineration. This method has been suggested previously, although it has not become popular. It is a very sensitive means of demonstrating the presence of iron in tissues and has application in cases in which the quantities of iron are very small or in which the iron is in a protein-bound form that fails to give a positive reaction with the usual Prussian blue technique.
THE HISTOCHEMICAL VISUALIZATION OF PROTEIN-BOUND SULFHYDRYL GROUPS WITH AN AZOMERCURIAL REAGENT
Authors: ENGEL MILTON B. , ZERLOTTI EUGENIO
Published: April 2011

An azomercurial reagent, 4-( p-dimethylamino benzene azo) phenyl mercuric acetate has been adapted for the histochemical demonstration of protein bound sulfhydryl groups. Sections were made from freeze-dried tissues including various keratinized structures, muscle and some parenchyma. They were stained directly in an alcoholic glycine buffered solution of the azomercurial. High resolution and good contrast were insured by viewing the sections with monochromatic light at 430 mµ.

The method, which is based on the work of Horowitz and Klotz, appears to be highly specific. Tissues which are very low in cysteine or virtually cysteine-free, do not react, and pretreatment of thiol containing sections with N-ethylmaleimide, p-chloromercuribenzoate or H2O2 prevents staining. Visualization of disulfide linkages following reduction was only partially successful due mainly to loss of soluble materials. Preliminary microphotometric studies suggest that the method could be used for semi-quantitative determinations. In many keratinizing tissues, cells and intercellular cement underlying the keratinized structures contain a high concentration of protein-bound thiol dispersed homogeneously as granules, or in fibrous form.
ELECTRON HISTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASE ACTIVITY IN THE SMALL INTESTINE OF MOUSE
Authors: BARKA TIBOR
Published: April 2011

In formalin-fixed or glutaraldehyde-fixed frozen sections of mouse jejunum acid phosphatase activity is localized electron histochemically to a group of cytoplasmic bodies. These bodies are structurally heterogeneous, sometimes with internal membranous components. The common position for these structures is below the terminal web. Following oil-feeding the number of enzyme-carrying granules is decreased. Enzyme activity occasionally surrounds lipid droplets.
THE EFFECT OF HYDROGEN ION CONCENTRATION ON FLUORESCENT LABELLED ANTIBODIES
Authors: HIRAMOTO R. , BERNECKY J. , JURAND J. , HAMLIN M.
Published: April 2011

3 - Aminotetramethylrhodamine, lissamine rhodamine B (sulforhodamine B, B. C. 12145 National Aniline Division, Buffalo, New York), 5-dimethylaminonaphthalene-1-sulfonyl chloride and amino fluorescein were employed for conjugation of horse antirabbit gamma globulin for the detection of rabbit antibodies to rat kidney glomeruli. For comparative purposes labelled antibodies having comparable number of residues/mole protein were employed. The possibility of selectively quenching fluorescent labelled antibodies with change of the hydrogen ion concentration was investigated.

Quenching of the fluorescence for fluorescein could be accomplished by dropping the pH of the medium to 3; partial reactivation of fluorescence occurred when the medium was made alkaline. Fluorescein fluoresced well from pH 11 to 7; appreciable quenching occurred at pH 5 and 4. From pH 4 complete reactivation occurred when the medium was made alkaline. Tetramethylrhodamine fluoresced well from pH 11 to 4; some loss of fluorescence was seen at pH 3. Lissamine Rhodamine B fluoresced best at pH 7 and 6. 5-Dimethylaminonaphthalene-1-sulfonyl chloride was a poor label and did not give the intensity of the other dyes at all pH ranges. At pH 2 all showed irreversible quenching.

Fluorescein- and tetramethylrhodamine-labelled antibodies used simultaneously on a single kidney section showed yellow fluorescence when both the orange of tetramethylrhodamine and green of fluorescein reacted at the same antigenic sites. Changes in the hydrogen ion concentration can be employed to selectively quench the fluorescence of fluorescein but not the tetramethylrhodamine-labelled antibodies.
ACID PHOSPHATASE IN CARCINOMA OF THE PROSTATE IN MAN
Authors: PARKIN LAUREEN , BYLSMA GLENN , TORRE A. V. , DREW D. , MADDEN ROBERT J.
Published: April 2011

Quantitative histochemistry of the human prostate demonstrates that in microtome sections of equal protein content the acid phosphatase activity is proportional to the number of acini present. The occurrence of cancer in these acini results in a profound lowering of the acid phosphatase activity. We postulate that the normal cell that evolves slowly into the cancer cell and highly differentiated carcinoma subsequently becomes undifferentiated and metastasizes. Finally, it is suggested that some change in the deoxyribonucleic-ribonucleic acid complex initiated by hormonal imbalance results in the loss of certain normal physiological functions such as the production and secretion of acid phosphatase and if these losses do not lead to cell death, tumor formation results.
THE CYTOLOGIC DEMONSTRATION OF β-GLUCURONIDASE EMPLOYING NAPHTHOL AS-BI GLUCURONIDE AND HEXAZONIUM PARAROSANILIN; A PRELIMINARY REPORT
Authors: HAYASHI MASANDO , NAKAJIMA YASUO , FISHMAN WILLIAM H.
Published: April 2011

The preparation of a new substrate for β-glucuronidase, naphthol AS-BI β-d-glucuronide, has been described. The potassium salt of naphthol AS-BI and acetobromomethyl glucuronate were reacted in ethanol. Unreacted anilide was removed and deacetylation and deesterification were carried out with barium hydroxide. The barium salt of naphthol AS-BI glucuronide was separated and was then converted to free acid. The compound has an elemental analysis which agrees with the theoretical one.

It has also been possible to devise a satisfactory cytochemical technique for the in situ demonstration of β-glucuronidase employing sodium salt of naphthol AS-BI glucuronide and hexazonium pararosanilin. The optimal staining reaction was obtained with 0.25 mM substrate and 1.8 mM diazo reagent at pH 5.2 in 20 to 30 minutes at 37°C for rat liver and kidney. The brilliant red dye was visualized at the site of enzyme activity mostly as discrete granules. A brief discussion concerned the evaluation of the present method as a cytochemical technique for the demonstration of β-glucuronidase.
QUANTITATIVE CHARACTERIZATION OF A HISTOCHEMICAL ENZYME SYSTEM
Authors: HOPSU V. K. , MCMILLAN P. J.
Published: April 2011

The renal alkaline β-naphthyl phosphatase has been studied in four assay systems ranging from the biochemical assay (homogenates incubated with substrate in the absence of diazonium salt) to the histochemical assay (frozen sections incubated with substrate in the presence of the diazonium salt, fast violet B). Quantitative and kinetic analyses were carried out for the purpose of determining how several factors influence the histochemical assay.

It was found that the stabilized diazonium salts partially inhibit as well as alter the kinetic characteristics of the phosphatase reaction. The steady state is rapidly reached in the histochemical system so that no lag period is observed, indicating that the coupling reaction and the penetration of substrate into the section are rapid processes. However, the evidence indicates that, especially at low substrate concentrations, a steady state substrate concentration gradient in the section is established so that the concentration of substrate in the section is less than it is in the medium. Thus, high substrate concentrations are required to demonstrate the total enzymic activity in a tissue section. Studies of this nature would appear to be prerequisites to any attempts at photometric quantitation of enzymic histochemical reactivities in tissue sections or zymograms.
ARGENTAFFIN GRANULES IN TOAD KIDNEY TUBULES
Authors: MUKHERJI MRITYUNJOY , SEN PARIMALBIKAS
Published: April 2011

A considerable amount of argentaffin granules was localised in the proximal tubules of the toad kidney.

While they were found to give argentaffin, metal chelating and alkaline azo coupling reactions in keeping with the general characteristics of the chromaffin cells, the chromaffin and Gibbs reactions could not be obtained.

It is, however, peculiar that the granules gave some acid azo coupling reaction and benzylidene reaction even after formol fixation. Positive fluorescence in ultraviolet light and some other reactions were also obtained to indicate that the compound studied was a tryptamine derivative with a tertiary amine at the end of the side chain.
HISTOCHEMICAL DISTRIBUTION OF LYSOZYME ACTIVITY IN ORGANS OF NORMAL MICE AND RADIATION CHIMERAS
Authors: SPEECE A. J.
Published: April 2011

A method for determining the distribution of lysozyme activity of animal tissues in situ has been devised. This method was based on a microscopically visible change in the staining reaction of Micrococcus lysodeikticus after incubation with cryostat sections of fresh frozen tissue. The sections were superimposed on a specially prepared film of bacteria. The change in the bacteria, apparently proportional to the lysozyme activity of the apposed cells, was shown by staining with a mixture of Alcian blue and basic fuchsin. This also stained the animal tissue suitably for histologic examination.

Lysozyme distribution in the normal mouse can be summarized as follows:

(1) very active in polymorphonuclear leukocytes, Paneth cells, and hemopoietic elements of bone marrow

(2) moderately active in lymphocytes (large more active than small), lung (apparently homogeneous), proximal renal tubule (more than glomerulus), pancreatic acinar cells, stomach glands, and lamina propria of small intestine and colon

(3) little or no activity in macrophages (except in lung), megakaryocytes, brain, liver, thymus, thyroid, adrenal, salivary gland, testis, upper epithelium of gastrointestinal tract, nor smooth, skeletal nor cardiac muscle.

Changes in lysozyme activity in the irradiated mouse at 9 days may be summarized as follows. There was an increase in lysozyme activity in the proximal tubules and glomeruli of the kidney. Acinar cells of the pancreas lost most of their activity. In the radiation chimeras the polymorphonuclear (PMN) leukocytes showed an increase in individual activity as well as increased numbers over normal. There was increased activity in the lamina propria of the gastrointestinal tract. All other changes noted could probably be attributed to the transplantation and proliferation of foreign cells and redistribution of the cellular elements of the host animals.

Acid phosphatase activity was determined for comparison, in the same tissues, with lysozyme distribution. Acid phosphatase activity was high in macrophages, acinar cells of liver and salivary glands, villus epithelium of small intestine, proximal tubules and glomeruli of kidney. These showed apparently constant acid phosphatase activity in the normal and treated animals. Whole organ changes seemed correlated with modified cell distribution.
HISTO- AND CYTOCHEMICAL LOCALIZATION OF DEHYDROGENASES IN RABBIT LENS EPITHELIUM
Authors: COTLIER EDWARD
Published: April 2011

Dehydrogenase distributions were determined with Nitro BT in fresh flat mounts of the rabbit lens epithelium and capsule. The capsule was unreactive and the epithelium displayed high lactic and malic dehydrogenase activities, and minimal uridine diphosphate glucose and glucose-6-phosphate dehydrogenase activities. In addition, lactic and malic dehydrogenases were present in higher amounts in the peripheral (pre-equatorial and equatorial) than in the central areas of the epithelium. Lactic dehydrogenase activity was specifically inhibited by sodium and potassium iodide, oxalic acid and sodium pyruvate, the latter effect reversed with cyanide. High lactic dehydrogenase activity in lens epithelium correlates adequately with previous microchemical determinations and points to a possible role of the epithelium in the active lactate production of the lens.
A METHOD FOR THE CYTOPHOTOMETRIC ESTIMATION OF NUCLEIC ACIDS USING METHYLENE BLUE
Authors: DEITCH ARLINE D.
Published: April 2011

When methylene blue is used to stains fixed tissue after acetylating to block competing protein basic groups, the amount of dye bound per cell reaches a limit largely independent of staining time, temperature and dye concentration and to a lesser extent pH. After extracting the nucleic acids, the tissue is unstained except for acid polysaccharide-containing areas. The intensity of staining does not depend upon alcoholic differentiation and the dye is used as a progressive stain. The amount of dye bound is stoichiometric with nucleic acid concentration. This is demonstrated by comparing the average dye binding to the nucleic acid concentrations in gelatin-nucleic acid model slides and for isolated HeLa nuclei. Although methylene blue is a metachromatic dye, analysis of absorption curves show that there is no significant differences in curve shape when the dye is bound either to tissue ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Hence total dye binding of nuclear chromatin may be used as an index of dye bound to both DNA and RNA. RNA may be extracted by a short hydrolysis in 1 N HCl; dye binding after such extraction represents dye bound to DNA. The amount of dye bound to chromosomal RNA can be estimated by the difference between these two figures and DNA/RNA ratios for methylene blue-stained chromatin can be determined in this manner.
HISTOCHEMICAL LOCALIZATION OF CERTAIN OXIDATIVE ENZYMES IN THE RAT TESTIS
Authors: AMBADKAR P. M. , GEORGE J. C.
Published: April 2011

The localization and distribution of β-hydroxybutyrate dehydrogenase, succinic dehydrogenase, malic dehydrogenase, and lactic dehydrogenase in the rat testis have been studied histochemically. Intense enzyme activity in the interstitium as well as tubules was observed. However, malic dehydrogenase was found to be more active in the interstitium than in the tubules. The distribution pattern of enzyme activity appeared in four different phases in the seminiferous tubules corresponding to the gradient in the spermatogenetic wave, thereby indicating a metabolic adaptation at subcellular level.
THE DEMONSTRATION AND DISTRIBUTION OF WATER SOLUBLE BLOOD GROUP O (H) ANTIGEN IN TISSUE SECTIONS USING A FLUORESCEIN LABELLED EXTRACT OF ULEX EUROPEUS SEED
Authors: KENT SIDNEY P.
Published: April 2011

A fluorescein labelled extract of Ulex europeus seed was found to be satisfactory for demonstrating water soluble H antigen in formalin fixed paraffin embedded tissue sections. Commercially available anti-A typing serum was labelled with fluorescein and used to demonstrate water soluble A antigen in formalin fixed paraffin embedded tissues. The reactivity of A and H antigen after neutral buffered formalin fixation was superior to that observed after Zenker's, Helly's or Bouin's fixation; reactivity in paraffin sections of formalin fixed tissues were equal to that of fresh frozen sections. Autolysis did not prevent the demonstration of blood group antigen A or H in fixed paraffin embedded tissues but was associated with some diffusion of the antigens.

The distribution of H antigen in formalin fixed paraffin embedded tissue section for patients with type A, B and O blood also was studied. In secretors blood group substances were widely distributed in the epithelial mucins of the body whereas in nonsecretors the antigens are largely confined to the deep portion of the pyloric mucosa of the stomach and Brunner's glands. The distribution of A and H antigen within a gland, such as Brunner's glands, was not homogeneous. That is, some acini contained A antigen but no H antigen; others contained H antigen but no A antigen: a few contained both A and H antigen. The significance of this mosaic distribution which previously has not been described was discussed.
PHOTOGRAPHIC CYTOPHOTOMETRY WITH A DUAL-MICROSCOPE. II. MICROSCOPE ASSEMBLY AND FILM-DYE EXTRACTIONS
Authors: KELLY JOHN W. , CLABAUGH W. A. , HAWKINS H. K.
Published: April 2011

Improved apparatus for dual-microscope photographic cytophotometry is described. The major component is a new comparison microscope, available commercially, whose binocular system is optically corrected for performance equivalent to that expected of one microscope. While current applications involve only standard bright-field and interference optics for absorption and dry-mass studies, a feature of the assembly is ready interchangeability of optics for fluorescence, polarization, phase, and dark-field microscopy.

A method for extraction of color-film dyes into dimethylsulfoxide is reported. The dye solutions are used for spectrophotometric measurement of amount of dye in a given area of film and, indirectly, for estimating the amount of chromophore in the corresponding area of a microscopic object.
INCORPORATION BY THE KIDNEY OF FLUORESCENT PITUITARY HORMONES
Authors: VILAR O. , ALVAREZ B. , DAVIDSON O. , MANCINI R. E.
Published: April 2011

Several Armour and NIH pituitary hormone preparations as well as other homologous serum protein fractions were labeled with fluorescent dyes (sulphorhodamine B, C.I. 45100, and fluorescein isothiocyanate). Rats were injected intravenously with the different labeled proteins and the animals killed at various intervals from 3 minutes to 10 days. Sections of the kidneys were studied under ultraviolet light (3,900 Å). It was observed that all the labeled proteins passed the glomerular filter and were accumulated by the cells of the proximal convoluted tubules. Labeled pituitary hormones were, in addition, reabsorbed by the thin limb and collecting tubules and appeared in the lumen of bladder. Filtration and reabsorption of proteins by the kidney can proceed independently of their nature and origin. All the proteins were reabsorbed with the same sequence, but pituitary hormones appeared faster and in larger amounts in the same sites of the nephron. They were also detected in the lumen of the ureter and in the bladder. It is assumed that the molecular weight of the proteins may play an important role, since the hormones' molecules were significantly smaller. It is remarkable that although the different hormones showed a preferential accumulation in the vessels of the corresponding target glands, they behave similarly in the kidney.
MORPHOLOGICAL AND CYTOCHEMICAL PROPERTIES OF THE HOLOCRINE CELLS IN THE EPIDIDYMIS OF THE MOUSE
Authors: MARTAN JAN , ALLEN JOHN M.
Published: April 2011

Holocrine secretory cells have been identified in the epithelium of the epididymal canal of the mouse. These cells develop from basal cells. During their differentiation they grow toward the lumen of the epididymal canal and come to form club-shaped structures with an expanded apical portion, a central nucleus and a thin stalk-like connection to the basement membrane. Mature holocrine cells are characterized by their high acid phosphatase and aliesterase activity. They also are highly active for succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase, and nicotinamide adenine dinucleotide phosphate diaphorase. Nucleoside diphosphatase, thiamine pyrophosphatase, adenosine triphosphatase, and alkaline nucleoside phosphatase are also found in these cells. These cells are also characterized by their reactivity with the Aoyama and periodic acid-Schiff reactions. They react moderately with the molybdate and Luxol Fast Blue MBS reactions for choline containing compounds. Mature holocrine cells may disintegrate in situ or may be discharged in toto into the lumen of the epididymal canal. Glycerylphosphorylcholine was identified in extracts prepared from sperm-free epididymides of mice. Glycerylphosphorylcholine reacts with Aoyama and periodic acid-Schiff reactions as do mature holocrine cells. This fact coupled with the identification of choline containing material in holocrine cells suggests that they may be one site for the formation of glycerylphosphorylcholine.
PRESERVATION OF TOTAL, LYO-, AND DESMOENZYME ACTIVITY IN MAMMALIAN SALIVARY GLANDS FOLLOWING CHLORAL HYDRATE FORMALIN FIXATION
Authors: CHAUNCEY HOWARD H. , KRONMAN JOSEPH H. , LEVITT MAYER A.
Published: April 2011

Acid phosphatase, alkaline phosphatase, nonspecific esterase, pseudocholinesterase, leucine aminopeptidase, and β-d-galactosidase activities of parotid and submandibular salivary glands were evaluated in six animal species. Assays for total enzyme, lyoenzyme, and demoenzyme were performed on unfixed and chloral hydrate formalin (CHF) fixed frozen cryostat sections.

Following fixation lyoenzyme (soluble fraction) activity exhibited extreme reduction for all enzymes except pseudocholinesterase, while the demoenzyme (insoluble fraction) activity of acid phosphatase, nonspecific esterase, pseudocholinesterase, and β-d-galactosidase was maintained at a high level or increased. Since removal of lyoenzyme activity and retention of desmoenzyme activity are requisites in histochemical localization, this fixative may be the preferred method for handling tissues prior to the localization of the above cited enzymes.

Several hypotheses are proposed to explain the increased desmoenzyme (nonspecific esterase and β-d-galactosidase) noted subsequent to fixation.
AUTORADIOGRAPHIC LOCALIZATION OF LIPID- AND WATER-SOLUBLE COMPOUNDS: A NEW APPROACH
Authors: WILSKE KENNETH R. , ROSS RUSSELL , Caldwell Leslie E. , Bockus Dawn E.
Published: April 2011

An approach to the localization of isotopically labelled lipid-soluble and water-soluble substances by autoradiography is presented. Previous attempts to retain such substances in situ in tissues have often been unsuccessful due to diffusion in the media involved in tissue preparation; and at the interface between the emulsion and the tissue section. Rats were given estradiol-H3 and aspirin-H3 as representatives of lipid-soluble and partially water-soluble compounds. After removal, tissues were quick frozen, frozen-dried, fixed in the vapor phase with osmium tetroxide or paraformaldehyde and vacuum embedded in Epon. One-micron sections were coated with emulsion and subsequently developed. Discrete localization of isotope was seen in autoradiographs. Data is presented to demonstrate the marked solubility of these compounds in the media used for routine fixation and dehydration, in contrast to their insolubility in epoxy resin. These findings present an opportunity for the cellular localization of numerous compounds by autoradiography that heretofore has not been possible.
CHARACTERIZATION OF ENZYMES HYDROLYZING ACYL NAPHTHYLAMIDES: II. TRIHALOGEN DERIVATIVES
Authors: HOPSU V. K. , SANTTI R. S. , GLENNER G. G.
Published: April 2011

Enzymes in guinea pig homogenates hydrolyzing a variety of halogenated and nonhalogenated acyl α- and β-naphthylamide substrates can be separated into two major groups on the basis of substrate hydrolysis rates, solubility and affector characteristics. Both groups of enzymes, those acting on the non-, mono- and dihalogenated acyl naphthylamides and those acting on trihalogen derivatives, have characteristics like those of arylesterases and are inseparable from enzymes hydrolyzing naphthol AS acetate. These enzymes are, However, separable on fractionation by starch gel electrophoresis and DEAE-cellulose chromatography from several enzymes catalyzing the hydrolysis of N-acyl amino acids (acylase I and II) and several amino acid β-naphthylamides. Species differences exist in the characteristics of enzymic hydrolysis of these acylarylamides.
QUANTITATIVE TESTS OF HISTOCHEMICAL METHODS FOR PHOSPHOMONOESTERASES IN BRAIN TISSUE
Authors: FRIEDE REINHARD L. , KNOLLER MECHTHILDE
Published: April 2011

Reliability of quantitative simultaneous coupling azo dye-based histochemical methods for acid and alkaline phosphatase activity was tested by comparing them with biochemical assays of tissue homogenates for the same activity using a modification of Gomori's phenol method. Gradations of histochemical staining observed with those methods, under carefully standardized conditions, reflected accurately enzyme levels obtained by assays of homogenized tissue.

Stability of dyes formed by the histochemical reaction and the optimal method of extraction were investigated. Both nonspecific absorption and lipid solubility of reaction product could be excluded as possible sources of artefacts. The influence of section thickness, number, and storage, volume of medium, incubation time, and formalin fixation on activity measurements was studied.
SELECTIVE STAINING OF COLLAGEN AND ELASTIN BY LUXOL FAST BLUE G IN METHANOL: A HISTOCHEMICAL STUDY
Authors: SALTHOUSE T. N.
Published: April 2011

Luxol fast blue G in saturated methanol solution selectively stained collagen and elastin in formalin-fixed tissue sections. This selective staining did not take place when a number of other solvents were used.

The possible mechanism of dye affinity for collagen and elastin was investigated. Deamination, acetylation, sulfation, potassium permanganate oxidation, and saponification did not greatly affect staining of collagen, although after deamination, and permanganate oxidation elastin did not stain. Therefore it is unlikely that chemical groups affected by these treatments play a significant role in collagen staining by this dye. Tannic acid treatment of sections prevented their staining by the Luxol-methanol solution. Similar treatment in vitro of collagen and elastin reduced staining but did not completely abolish it. Tannic acid tanning of collagen affects its physical properties by introducing cross linkages, and also interacts with basic protein groups. Nonionic groups are also blocked. Both the physical properties of collagen as a polymer, and its chemical properties may be involved in its affinity for Luxol fast blue G in methanol. Luxol fast blue G is a di- oItolylguanidine salt of a trisazo dye. It is possible that only the molecular arrangement of the dye in methanol is able to complex with collagen. After many of the blockade treatments elastin did not stain. The groups blocked by these treatments must be necessary for binding of the dye by elastin, but not for collagen.

The method is simple and would seem adaptable to routine histological use and for studies of the histochemical and staining properties of collagen and elastin.
A ONE-WAVELENGTH, TWO-AREA METHOD IN MICROSPECTROPHOTOMETRY FOR PURE AMPLITUDE OBJECTS
Authors: GARCIA ALFREDO MARIANO
Published: April 2011

A one-wavelength method for nuclei in smears is tested and discussed. It eliminates need for visual estimates of areas and measurements to check for possible slight variations in absorption spectrum which would vitiate applicability of the two-wavelength method. Extinction ( E1) is obtained from an intranuclear plug measurement; area of absorbing material is computed as a function of ratios between "one minus transmittances", one intranuclear, the second including in addition "free" or "non-absorbing" areas around it. If T1 represents intranuclear transmittance, T2 total transmittance (nucleus plus free area), A1 the area of the nucleus and A2 the area from which T2 was obtained (absorbing plus nonabsorbing media), total extinction times area is computed as

[See equation in the PDF file]
ADENOSINE TRIPHOSPHATASE ACTIVITY IN RAT BRAIN FOLLOWING DIFFERENTIAL FIXATION WITH FORMALDEHYDE, GLUTARALDEHYDE, AND HYDROXYADIPALDEHYDE
Authors: TORACK RICHARD M.
Published: April 2011

Differential fixation of rat brain has been described using formaldehyde, glutaraldehyde and hydroxyadipaldehyde by perfusion or prolonged immersion. Fixation by prolonged immersion appears preferable since it produces a similar result with greater simplicity. Distribution of reaction product resulting from adenosine triphosphate hydrolysis in these fixed brains appears different and characteristic for each of these fixatives when they are used in this manner. More adenosine triphosphatase activity in rat brain was observed following formalin fixation than after fixation with either glutaraldehyde on hydroxyadipaldehyde; in this respect formalin fixation is recommended for over-all study of adenosine triphosphatase activity in the brain. The use of glutaraldehyde and hydroxyadipaldehyde seems indicated when study of enzymes surviving these fixatives is desired. Beside varying inactivation by different aldehyde fixatives, adenosine triphosphatase activity of rat brain has been characterized by distinct substrate preference and by chemical inhibition.

Specificity of adenosine triphosphatase localization in rat cerebrum by electron microscopy seems enhanced by such differential fixation since differences in enzymatic activity not previously apparent can he recognized in closely related structures such as components of the blood-brain barrier. Since adenosine triphosphatase activity of astroglia in corpus callosum as well as in subpial and subependymal glial networks is glutaraldehyde resistant, an enzymatic similarity perhaps related to their physiological activity is indicated in these cells. Astroglia of cortex evince enzyme activity that survives only formalin fixation, suggesting a different function for cortical astrocytes. The enzymatic activity of oligodendrocytes appears to be a diphosphatase inactivated by glutaraldehyde and hydroxyadipaldehyde, and in this is strikingly similar to diphosphatase of Golgi apparatus.
A NOTE ON THE CHEMICAL MECHANISM OF THE NAUTA-GYGAX TECHNIQUE
Authors: GIOLLI ROLAND A.
Published: April 2011

This research attempts to determine which chemical groups are involved in Nauta impregnation of degenerating peripheral and optic nerve fibers. Before the Nauta-Gygax technique, tissues either were treated with bromine water to block unsaturated bonds or with acetone or chloroform-methanol to extract various lipid constituents.

Bromination completely suppressed Nauta impregnation of degenerating optic and sciatic nerve fibers. This, together with data provided by extracted preparations, implicates unsaturated lipids, probably cholesterol esters, in the impregnation. The basic similarity in chemical mechanism of the Nauta and Marchi impregnations of degenerating fibers is discussed. Whereas the Marchi reaction in nerve fibers is limited to degenerating myelin, data are presented indicating that Nauta impregnation involves both degenerating myelin sheath and axis cylinder.
SEPARATION AND RELATIVE QUANTITATION OF MOUSE PLASMA ESTERASES WITH DISC ELECTROPHORESIS
Authors: ALLEN R. C. , POPP R. A. , MOORE D. J.
Published: April 2011

A technique for the qualitative and quantitative study of mouse plasma esterases using disc electrophoresis in acrylamide gel is described. This technique has been employed to study substrate specificity and quantitative relationships among various mouse strain and sex dependent esterases. Properties of acrylamide gels are discussed relative to their effect on the quantitative estimation of plasma esterases.
COMPOSITION OF CEMENT LINES IN BONE
Authors: PHILIPSON B.
Published: April 2011

In cement lines and in adjacent Haversian systems from bone of orangutan and whale the content of mineral, organic matrix, and sulfur was determined microradiographically and the arrangement of the mineral part was studied by X-ray diffraction. Cement lines were often highly mineralized, maximum being 50% by volume. This value was a few per cent higher than the highest value for Haversian systems. Content of collagen varied to a certain extent, and low values were found in cement lines. Determinations of sulfur and X-ray diffraction studies of the organization of the mineral fraction did not reveal any significant differences between cement lines and adjacent osteons.
CORRELATED LIGHT AND ELECTRON MICROSCOPE OBSERVATIONS ON GLYCOPROTEIN-CONTAINING GLOBULES IN THE FOLLICULAR CELLS OF THE THYROID GLAND OF THE RAT
Authors: VAN HEYNINGEN HUBERTA E.
Published: April 2011

Two carbohydrate staining techniques were applied to sections of rat thyroid gland: periodic acid-silver methenamine to thin sections for electron microscopy, and periodic acid-Schiff to thick sections for light microscopy. The latter were compared with adjacent thin sections for identificatoin with the electron microscope. Two types of globules in thyroid follicular cells stained with both methods. Globules of the first type are relatively large (usually 0.5 to 3 µ) with electron opacity very similar to that of follicular colloid; when stained with toluidine blue they have the same gray shade as follicular colloid. These similarities suggest that their periodic acid reactivity is due to the same glycoprotein as that of follicular colloid, namely thyroglobulin, and that the origin of these "intracellular colloid droplets" is the colloid in the lumen. The second type comprises medium-sized (usually 0.1 to 1 µ) fairly electron opaque globules having fine particles (~70 Å) dispersed in their matrices and sometimes containing membrane fragments or other irregularities; when stained with toluidine blue these globules stand out dark blue. Although their periodic acid reactivity indicates that these globules also contain glycoprotein, their ultrastructure and staining characteristics suggest that their composition differs from colloid. It is possible that they represent enzyme or zymogen-containing granules. A third type of globule, which on account of its intense staining in some periodic acid silver methenamine preparations could perhaps also be periodic acid reactive, concerns small (usually 0.02 to 0.2 µ) globules, mainly accumulated beneath the apical border of the cell. These globules, known as "apical vesicles," are believed to contain the not-yet-iodinated precursor of thyroglobulin.
HISTOCHEMISTRY OF TISSUE CULTURE CELLS: A STUDY OF THE EFFECTS OF FREEZING AND OF SOME FIXATIVES
Authors: MAGGI V. , RIDDLE P. N.
Published: April 2011

It has been shown that a) the membranes of HeLa and L cell lysosomes are readily permeable to naphthol AS-AM phosphate when used as a substrate for acid phosphatase, b) freezing as well as fixation affect the permeability of the lysosomal membrane to both naphthol AS-AM phosphate and β-glycerophosphate, c) none of these treatments seem to affect greatly the plasma or the mitochondrial membranes and d) the distribution of phospholipids in the cells is also affected by freezing or fixation.
HISTOCHEMICAL STUDIES OF MITOCHONDRIAL VARIATION DURING AEROBIC GROWTH OF RESPIRATION-NORMAL BAKER'S YEAST
Authors: AVERS C. J. , LIN F. H. , PFEFFER C. R.
Published: April 2011

Histochemical localizations of cytochrome oxidase, succinic dehydrogenase, DPNH-tetrazolium reductase, and TPNH-tetrazolium reductase activities revealed at least two kinds of mitochondria in the intracellular population. The total chondriome in stationary phase cells contains about 45 mitochondria, all with cytochrome oxidase activity. But, only about 30 mitochondria per cell were active for dehydrogenase or reductases. The differences in mitochondrial enzyme activities persisted throughout the growth cycle, showing different numbers of active mitochondria and different rates of their increase and decrease for all four enzyme systems. Manometric data verified the differences between cytochrome oxidase and succinic dehydrogenase for the earlier phases of the growth cycle. In histochemical counts, zero values for all four enzymes occurred in late acceleration phase, but persisted into log phase only for the tetrazolium reductases. Both cytochrome oxidase- and succinic dehydrogenase-active mitochondria began to increase in numbers at the inception of log phase, but at very different rates. The demonstration of more than one kind of mitochondrion in the common nucleocytoplasmic system of a single cell was considered to be evidence of some measure of autonomous control of the mitochondrial phenotype.
HISTOCHEMICAL DEMONSTRATION OF N-ACETYL-β-GLUCOSAMINIDASE EMPLOYING NAPHTHOL AS-BI N-ACETYL-β-GLUCOSAMINIDE AS SUBSTRATE
Authors: HAYASHI MASANDO
Published: April 2011

The N-acetyl-β-glucosaminide of naphthol AS-BI (7-bromo-3-hydroxy-2-naphth- o-anisidide) was obtained by reacting the anisidide with acetochloroglucosamine in aqueous alkaline acetone. After removal of O-acetyl groups with methanolic ammonia and recrystallization from aqueous methanol, the compound may be used as a substrate for N-acetyl-β-glucosaminidase.

Formol-calcium fixed frozen sections were incubated with 0.5 mM substrate in the presence of hexazonium pararosanilin or fast garnet GBC at pH 5.2. Staining appeared in the cytoplasm mostly as discrete granules at the presumed site of enzyme activity and was inhibited in the presence of N-acetylglucosaminolactone. In the rat kidney a number of spherical granules which stained for the enzyme were observed in the proximal portion of the proximal convoluted tubules. In the liver staining was recognized as small granules localized at the pericanalicular cytoplasm of parenchymal hepatic cells. The general cytological localization in these tissues was quite similar to that of other lysosomal enzymes. The limitations of the technique were briefly discussed.
EXTRAVASCULAR AND INTRATUBULAR DIFFUSION OF LABELED SERUM PROTEINS IN THE RAT TESTIS
Authors: MANCINI R. E. , VILAR O. , ALVAREZ B. , SEIGUER A. C.
Published: April 2011

Rat whole serum, albumin, globulins, and fibrinogen were labeled with fluorescent dyes (sulforhodamine B. CI No. 45100 and fluorescein isothiocyanate); albumin was also tagged with radioactive iodine (I131) and tritium (H3). In addition heterologous albumin was also labeled with fluorescent dyes and radioiodine. The proteins were intravenously injected in prepubertal, pubertal and adult rats, and their decay in the circulation and histological distribution in the testis and epididymis was studied. As controls other animals were similarly injected with free labels alone and with labeled denatured and degraded albumin; also unlabeled homologous and heterologus albumins were administered followed by incubation of both organs with the corresponding labeled antisera applying the Coons' technique. It was observed: 1) with the exception of fibrinogen, labeled serum proteins rapidly appeared in the lumina of vessels, diffused extravascularly in the intertubular spaces and finally arrived inbetween the germinal cells and in the lumina of seminiferous tubules. Also labeled material was present in the lumen of epididymal canaliculi but not in the ductus deferens. 2) This extravascular and intratubular diffusion was parallel to the fast component of the time decline curve of labeled homologous serum proteins in the circulation. 3) There was no great difference between young and adult rats in the extravascular diffusion process, but intratubular passage was higher in pubertal and adult animals. 4) Control experiments revealed time presence of some fluorescent material only in the vessels and macrophages, whereas the immunofluorescent technique showed to very similar localization of unlabeled proteins to that provided by the injection of directly labeled proteins.
HISTOCHEMICAL STUDIES ON GLYCOSIDASES OF THE RAT OVARY AND PLACENTA
Authors: BULMER D.
Published: April 2011

In the rat ovary and placenta the distribution of β-glucuronidase and β-galactosidase activities shown by the 6-bromo-2-naphthol post-coupling methods differs markedly from the distribution of β-glucuronidase activity which is indicated by the Fishman-Baker ferric hydroxyquinoline technique. In the ovary the intense reactions in interstitial cells and luteal cells with the post-coupling methods may be partly due to diffusion of intermediate reaction product. In the placenta a distribution is indicated for both glycosidases which is similar to the distribution pattern of acid phosphatase and organophosphate-resistant esterase. It is unlikely that the granular reactions obtained in some situations with the post-coupling methods can be regarded as an exact cytological localization of enzyme activity.

The simultaneous coupling method for acetylglucosaminidase shows strong reactions in ovarian macrophages and atretic follicles, with weaker activity in corpora lutea, interstitial cells, and theca interna. In the placenta the visceral endoderm and decidua are strongly reactive, with weaker activity in the metrial gland and trophoblast. In some situations the reaction product is deposited in granular from, probably on the surface of lysosomal particles.
HISTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE IN HAMSTER AND RABBIT EGGS
Authors: ISHIDA K. , CHANG M. C.
Published: April 2011

Succinic dehydrogenase activity, using Nitro-blue tetrazolium salt as electron acceptor, has been demonstrated histochemically in hamster and rabbit eggs before implantation. The formazans formed as blue granules are spread throughout the cytoplasm and among vitelline granules, corresponding to the distribution of mitochondria. The intensity and distribution of these granules vary according to the developmental stage of the eggs. Incubation of hamster eggs for 1 hour produced a weekly and evenly distributed reaction in most (92%) of the unfertilized eggs, while 57% of fertilized 1-cell eggs showed a moderate reaction. Although 84% of the 2-cell eggs, 95% of the 4-cell eggs, and 98% of the 8-cell eggs showed moderate reaction, 78% of blastocysts showed strong activity. In the rabbit egg the time required to develop the reaction was about 4-6 hours for unfertilized and pronuclear eggs, 3-5 hours for 2-cell eggs, 2 hours for 32-cell eggs, 1-2 hours for morulae, and only 0.5-1 hour for blastocysts. The development of succinic dehydrogenase in mammalian eggs at the first cleavage, its gradual increase during cleavage, and its tremendous increase at the blastocyst stage have been clearly demonstrated. The importance of this enzyme activity in relation to oxygen uptake, pathways of carbohydrate metabolism, and the degeneration of eggs are discussed.
FACTORS AFFECTING ENZYMATIC REMOVAL OF GLYCOGEN FROM TISSUE SECTIONS AND FROM ENTIRE RAT LIVER BLOCKS
Authors: PAEGLE RONALD D.
Published: April 2011

The rates of enzymatic glycogen removal from two different types of rat liver preparations were determined histochemically, employing the periodic acid-Schiff method to visualize glycogen. The first preparation consisted of elongated liver blocks (approximately 2 x 2 x 10 mm) which were fixed, then exposed to the alpha amylases before dehydration and embedding. The second type of preparation consisted of 5-8 µ thick, routinely prepared sections. The glycogen was removed considerably slower from the formaldehyde fixed tissue blocks than from the sections. The rate of glycogen hydrolysis was enchanced in the liver blocks by brief periods of autolysis, by removal of lipids and by washing before exposure to the enzyme.

Hydrolysis by amylase was slower when 0.18 M phosphate was used with the formaldehyde during fixation than when veronal acetate or 0.018 M phosphate buffers were used at the same pH 7.2-7.4. Many peripherally located cells retained glycogen even after incubation of the entire blocks for 24 hours in alpha amylase when the buffer during fixation was 0.18 M sodium Phosphate.

The contaminating ribonuclease present in malt diastase preparations penetrated readily into the cells of the elongated liver blocks but the protease affected only the periphery during the 24 hour incubation.

The differences in resistance to enzymatic hydrolysis were not confined to the tissue blocks. The type of buffer and fixative used for tissue fixation determined the subsequent susceptibility of glycogen to hydrolysis in sections also. The list is arranged in order of progressively decreasing resistance to enzymatic hydrolysis: 3.8% formaldehyde or 3% glutaraldehyde with 0.18 M phosphate, 3.8% formaldehyde with 0.018 M phosphate or 0.18 M veronal acetate, 3% glutaraldehyde with distilled water, 3.8% formaldehyde with 0.129 M veronal acetate or distilled water, and 70-95% ethanol. In addition, glycogen was removed slower from the peripheral zones of the sections than from the central portions.