PROTON-INDUCED X-RAY ANALYSIS OF TRACE ELEMENTS IN TISSUE SECTIONSJUNDT, F. C.; PURSER, K. H.; KUBO, H.; SCHENK, E. A.
doi: 10.1177/22.1.1pmid: 4596692
A 3-meV proton beam from the MP Van de Graaff accelerator at the Nuclear Structure Research Laboratory of the University of Rochester has been used to induce characteristic x-rays of trace elements from a variety of biologic samples. The x-ray spectra have been measured with a high resolution Si(Li) detector which can detect and separate in energy x-rays from neighboring elements with Z > 13. Tissue homogenates, frozen sections and formalin-fixed, paraffin-embedded sections of various tissues obtained from surgical and autopsy specimens have been analyzed. Measurements have been made to check the sensitivity and reproducibility of the method. Data accumulated thus far indicate that this method is useful in evaluating absolute concentration of elements usually present in a given tissue and detecting environmental elements which may have accumulated in a given tissue.
CYTOCHEMICAL LOCALIZATION OF BRAIN NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED)-DEPENDENT DEHYDROGENASES QUALITATIVE AND QUANTITATIVE DISTRIBUTIONSSIMS, K. L.; KAUFFMAN, F. C.; JOHNSON, E. C.; PICKEL, V. M.
doi: 10.1177/22.1.7pmid: 4133072
This study compares the histochemical and microchemical localizations of nicotinamide adenine dinucleotide phosphate (reduced) and nicotinamide adenine dinucleotide (reduced) diaphorases and four nicotinamide adenine dinucleotide phosphate (oxidized)-dependent enzymes (glucose 6-phosphate, 6-phosphogluconate, malate and isocitrate dehydrogenases) in areas of rat metencephalon and spinal cord. For the four nicotinamide adenine dinucleotide phosphate (NADP) enzymes, the pattern of localization following use of a modified tetrazolium procedure was compared with quantitative data obtained by microdissection from the same areas in adjacent sections. Optimal experimental conditions for reaction pH, temperature, substrate, cofactor and divalent cation concentrations were used for both the quantitative analysis following microdissection and the histochemical tetrazolium procedure. Consecutive sections were also examined for isocitrate dehydrogenase (nicotinamide adenine dinucleotide (oxidized)) and nicotinamide adenine dinucleotide (reduced) diaphorase activities in addition to seriatim thionine reference sections. Our results indicate that, within the central nervous system, certain characteristic qualitative differences exist in the distribution of the nicotinamide adenine dinucleotide phosphate (oxidized)- and nicotinamide adenine dinucleotide (oxidized)-dependent dehydrogenase enzymes. Nicotinamide adenine, dinucleotide enzymes are visualized predominantly in neuronal cell bodies or neuropil consisting primarily of neuronal processes; in adjacent sections, NADP enzyme activities are visualized almost exclusively in glial elements with two important exceptions. The first is the cerebellar molecular layer where the results from both micro- and histochemical techniques indicate high levels of the NADP enzymes relative to other dehydrogenases and high activity relative to the levels of these NADP enzymes in other nervous system areas. The second exception occurs in those neuronal groups known to contain high levels of catecholamines; these data are the subject of a companion report.
LOCALIZATION OF NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE-DEPENDENT DEHYDROGENASES IN CATECHOLAMINE-CONTAINING NEURONS OF RAT BRAIN STUDIES ON THE NUCLEUS LOCUS CERULEUSKAUFFMAN, F. C.; BLOOM, F. E.; SIMS, K. L.; PICKEL, V. M.
doi: 10.1177/22.1.20pmid: 4133068
Histochemical and cytochemical methods have been used to localize glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate (nicotinamide adenine dinucleotide phosphate (NADP)) dehydrogenase and malic enzyme in the nucleus locus ceruleus and other catecholamine-containing neuronal groups of the rat brain stem. The four NADP-dependent dehydrogenases were studied by both quantitative and qualitative histochemical techniques using adjacent tissue sections. Both types of analyses were done on neuronal nuclei known to contain catecholamines in high concentration, the nucleus locus ceruleus and the superior cervical sympathetic ganglion; other known catecholamine-containing nuclei were surveyed by the cytologic technique only. There was intense staining of cell bodies and neuropil of the nucleus locus ceruleus after all four of the NADP dehydrogenase histochemical reactions. In contrast, little staining was observed in the adjacent vestibular nuclei or mesencephalic root nucleus of the trigeminal with the exception of appreciable glucose 6-phosphate dehydrogenase activity present in neuropil elements. Quantitative microchemical determinations in the nucleus locus ceruleus corroborate the histochemical results which indicated high NADP dehydrogenase activities particularly for 6-phosphogluconate dehydrogenase. This same pattern of high NADP enzyme activity as determined by combined cytochemical and quantitative chemical techniques was also observed in the superior cervical ganglion and cytochemically in other catecholamine-containing nuclei of the brain stem. Our findings suggest that a high capacity to generate or utilize nicotinamide adenine dinucleotide phosphate (reduced) may be characteristic of those neurons which either receive adrenergic terminals or synthesize catecholamines.
DIETHYLENE GLYCOL DISTEARATE AS AN EMBEDDING MEDIUM FOR HIGH RESOLUTION LIGHT MICROSCOPYTALEPOROS, PLATO
doi: 10.1177/22.1.29pmid: 4596693
Owing to its physical properties, diethylene glycol distearate is especially suited for obtaining thin sections of high quality with little or no compression. Tissues can be cut as thin as 1 µm on a steel knife without cooling, but a water trough is essential. The wax is translucent, so that specimens can be oriented for a desired plane of sectioning. The wax is also of sufficient hardness to serve as an embedding medium for osmium-fixed material and can readily be removed from such tissue sections for rapid phase contrast studies. A general method is described for embedding and sectioning with diethylene glycol distearate wax and examples of freeze-substituted and osmium-fixed material are illustrated. It is concluded that this synthetic wax can offer a convenient and simple means for obtaining thin sections for most aspects of high resolution light microscopy.
HUMAN LEUKEMIC CELLS CYTOCHEMICAL STUDIES ON ACIDIC NUCLEAR PROTEINSDESAI, L. S.; FOLEY, G. E.
doi: 10.1177/22.1.40pmid: 4133069
Utilizing staining with alkaline fast green (pH 8.1) and toluidine blue O (pH 9.0) stains, the nuclear basic and acidic protein content of individual human lymphocytic cells has been quantitated cytochemically with high resolution, rapid scanning instruments. There were distinct differences in the distribution patterns of the acidic and basic proteins in these human lymphocytic cells. Nucleoli were densely stained by toluidine blue O, confirming the presence of acidic proteins. The acidic nuclear proteins:DNA and basic nuclear proteins:DNA ratios are consistent and quantitatively similar, irrespective of the diagnostic category from which the cells derived and there was a definite correlation between nuclear proteins and DNA content. However, the acidic nuclear proteins represented a larger proportion of the total dry mass of the cell than did the basic nuclear proteins, an observation which may prove to be of interest with respect to control mechanisms.
CYTOCHEMICAL DEMONSTRATION OF HISTONASE ACTIVITY IN HUMAN LEUKOCYTESKASS, LAWRENCE; SCHNITZER, BERTRAM
doi: 10.1177/22.1.46pmid: 4133070
A cytochemical technique was devised for demonstrating a lysosomal enzyme capable of hydrolyzing histone. The test for histonase involved incubation of peripheral blood leukocytes in a mixture of calf thymus histone (substrate) and Fe(NH4)2(SO4)2·6H2O. Combination of this iron salt occurred with products of histone cleavage (arginine, lysine, glycine). The iron-amino acid complex was made visible in the cytoplasm of the cell by the Prussian blue stain. Histonase enzyme was found to have a narrow substrate specificity and to react only with histone and not with a variety of biologic and synthetic substrates for protease activity. The role of histonase in the transfer of information from nucleus to cytoplasm is discussed.