bluuebunny/crossref_metadata_2025_split huggingface dataset:
A QUANTITATIVE APPRAISAL OF ENZYME HISTOCHEMICAL METHODS IN BRAIN TISSUE
Authors: FRIEDE REINHARD L. , FLEMING LADONA M. , KNOLLER MECHTHILDE 
Published: April 2011

The quantitative reliability of the histochemical methods for diphosphopyridine nucleotidediaphorase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, monoamine oxidase, and lactic dehydrogenase in brain tissue was tested by comparison with enzyme assays in tissue homogenates. The reaction for diphosphopyridine nucleotidediaphorase in formalin-fixed tissue can be considered a microchemical method; it adequately reflects the gradations among nuclei measured by assays in fresh tissue homogenates. The same was true for the reaction for succinic dehydrogenase in unfixed tissue sections. In general, however, enzyme histochemistry in unfixed sections was subject to substantial error due to leakage of enzyme activity into the incubation medium (or washing fluid) . This leakage was prevented by fixation of the tissue if fixation was compatible with the enzyme. The extent of leakage varied greatly among regions of a given section; neuropil generally leaked more than cell bodies. The rate of leakage also differed markedly among enzymes and was influenced by a variety of physical factors such as thickness of sections, storage, washing, etc. Thin cryostat sections were particularly susceptible to leakage. Working standards for the quantitative use of enzyme histochemical methods in brain tissue were developed. A combination of assay of homogenates with histochemistry is recommended, whereby the relative significance attributed to either method must be determined separately for every individual enzyme.
BASIC FUCHSIN AND THE FEULGEN REACTION: SIGNIFICANCE OF THE DYE FOR CYTOPHOTOMETRIC DETERMINATION OF DEOXYRIBONUCLEIC ACID IN CELL NUCLEI
Authors: LODIN Z. , MÜLLER J. , PILNÝ J. , HARTMAN J. 
Published: April 2011

The effect of basic fuchsins of different origin on the final pattern of extinction of the Feulgen reaction was determined cytophotometrically. The following products were compared: basic fuchsin by Light, by National Aniline (C.I. No. 42510 (formerly 677)), Lachema, Harleco, (Cert. no. LF 23, 99% dye content), and acridine-free pararosaniline, of Bayer. The low dye content of basic fuchsin and the admixture of other coloured components results in a decrease of the extinction values obtained cytophotometrically and in a decrease in interpretability of nuclear histograms.
DEVELOPMENT OF A HISTOCHEMICAL METHOD FOR α-d-GALACTOSIDASE AND ITS DISTRIBUTION IN THE RAT
Authors: MONIS BENITO , TSOU KWAN-CHUNG , SELIGMAN ARNOLD M. 
Published: April 2011

A histochemical method for the demonstration of α-d-galactosidase activity in formalinfixed, frozen sections was developed by the synthesis of 6-bromo-2-naphthyl-α-d-galactopyranoside. The histochemical procedure is similar to that developed for other glycosidases and utilizes the post-incubation coupling principle. A survey of the organs of the rat revealed the highest activity in the cytoplasm of epithelial cells of Brunner's glands of the intestine, distal segment of proximal tubules of the kidney, thyroid and parathyroid glands and very strikingly in heterophiles and megakaryocytes.
FIXATION OF CELLS AND TISSUES BY CHLORO-S-TRIAZINES
Authors: GOLAND PHILIP , ENGEL MILTON 
Published: April 2011

Cyanuric chloride and related water-soluble dichloro-s-triazines (Procion dyes and Lissatan PR) or monochloro-s-triazines (Procion H and Cibacron dyes, were utilized as fixatives for fresh and freeze-dried animal tissues. With these reagents, endogenous and exogenous substances were irreversibly insolubilized and rendered resistant to chemical and enzymatic degradation. Certain components have been preserved heretofore lost by other methods. Fixation may depend mainly on the formation of covalent and chelate bonds with the tissue colloids. These chloro-s-triazines can be applied in various ways to meet specific fixation needs. In order to avoid the effects of solvents, cells and thin segments of tissues can be cyanurated in the vapors of cyanuric chloride. The water-soluble Procion dyes and Lissatan PR are effective fixatives for some constituents in fresh tissues; enamel matrix is especially well preserved. The most effective of these cyanurating agents for general use appears to be cyanuric chloride in nonpolar solvents when applied to freeze-dried tissues. Following some cyanurations, unreacted striazine halogen appears available for further condensation with active hydrogen in reactants containing certain functional groups (i.e.,—NH2, —SH, —OH, etc.) By condensations with suitable reactants it appears possible to establish crosslinked polymers in tissues. There is evidence that reaction of fresh hydrated tissues with dichloro-s-triazines imparted some water-repellency to the tissues. Through unreacted halogen in the previously condensed s-triazinyl rings, it appears possible at ambient temperature to effect additional condensations with water-soluble agents utilized in hydrophobing treatments.
PERCHLORIC ACID EXTRACTION AS A HISTOCHEMICAL TECHNIQUE
Authors: ALDRIDGE WILLIAM G. , WATSON MICHAEL L. 
Published: April 2011

Investigations have been carried out to determine the efficiency of cold perchloric acid in the differential extraction of RNA from tissues fixed in acrolein and Carnoy's fixative. It has been found that this reagent will selectively extract RNA from tissue without extracting or causing significant depolymerization of DNA. The DNA in the tissue is, however, denatured (i.e., its secondary structure is destroyed) and depurinized to a degree dependent upon the time of extraction. When the procedure was carried out at 4°C, there was no evidence of extraction of proteins, glycoproteins, or hipoproteins. Some phospholipid was extracted under these conditions. It is concluded that the cold perchloric acid technique for differential extraction of RNA from fixed tissues is useful and not subject to the faults often ascribed to it in the histochemical literature. The significance of loss of Feulgen and methyl green staining following prolonged PCA extractions is discussed in relation to the chemistry involved in DNA-methyl green binding and the Feulgen reaction. Abbreviations used: Perchloric acid = PCA Desoxyribonucleic acid = DNA Ribonucleic acid = RNA Trichloroacetic acid = TCA Thymine/adenine ratio = T/A Cytosine/guanine ratio = C/G Optical density = OD
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.
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 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.
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.
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.
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.
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.
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.