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doi: 10.1177/23.3.1168665pmid: 1168665
Two polysaccharides, dextran 250 and dextran 70, were covalently linked to antibody molecules, antihuman immunoglobulin G and antihuman type O red blood cells. In electron microscope preparations exposed to lead citrate, polysaccharides, because they chelate lead, were quite dense. Polysaccharides served as a tag for the antibody molecules. Also, bacterial dextran 1355 was used to demonstrate antibody molecules on the surface of ascites tumor cells which are known to be producing a specific antibody to bacterial dextran 1355. The varying sized polysaccharide molecules that are readily available commercially, the high electron density of the polysaccharides after lead staining and a mild procedure for covalently linking polysaccharide to antibody make polysaccharides attractive as particulate labels for antibody in electronmicroscopy.
doi: 10.1177/23.3.236340pmid: 236340
Colloidal alpha-stannic acid and a negative iron colloid obtained from ferric hydroxide and potassium ferrocyanide, both negative sols being stable within a wide pH range, were refined as surface protein electron markers. Because of the relatively small size of its particles, colloidal alpha-stannic acid was used for staining all surface proteins. According to the pH at which the negative iron colloid was applied, it revealed either all surface proteins, or because of its large colloidal particles, stained basic proteins. This differential staining capability of the iron colloidal has been demonstrated previously on various control preparations (Puvion E, Blanquet PR: J Microsc 12:171, 1971). Controls on the affinity of the two colloids to surface amino groups were carried out on rat liver, mouse fibroblasts, HeLa and KB cells, Ehrlich and Zajdela ascites cells subjected to prior enzymatic and chemical treatments (incubation with neuraminidase or phospholipase C, esterification, acetylation or lipid extraction). At any pH below 9, the two sols stained proteins in the outer hydrophilic leaflet of esterified cells with relative selectivity, but the alpha-stannic acid showed them more accurately. The iron sol did reveal at high pH protein components of high isoionic point on the surfaces of rat hepatocytes and ascites cells which had only been treated with neuraminidase.
doi: 10.1177/23.3.1092753pmid: 1092753
A micromethod was employed to estimate quantitatively and reproducibly the deoxyribonucleic acid (DNA) content of isolated rat islets of Langerhans. The DNA content per islet varied linearly with the mean diameter or the dry weight of the islets isolated. The DNA in the freeze-dried islets of male Sprague-Dawley or Wistar rats was about 21.0 mug/mug islet dry weight. Three to four weeks after hypophysectomy, with or without short term bovine growth hormone replacement, the DNA content per unit dry weight of islets was not significantly altered. Islet DNA content and islet dry weight are proposed as an interconvertible and reliable basis of reference for measurements of different islet functions.
doi: 10.1177/23.3.47868pmid: 47868
A method for light microscopic localization of adenylate deaminase in sections of frozen rat quadriceps muscle is described. The method depends on the hydrolysis of 6-chloropurine ribonucleotide (the 6-chloroanalogue of adenylate) and the trapping of Cl minus by Ag plus. The resulting AgCl precipitate was made visible by exposing the sections to light. After this treatment black deposits about 1 mu in diameter were seen in muscle cells. These observations indicate that adenylate deaminase of rat quadriceps muscle is located at discrete sites within the muscle cells.
doi: 10.1177/23.3.47869pmid: 47869
In this study we compared horseradish peroxidase (HRP)-labeled rabbit antihuman immunoglobulin G (IgG) conjugates, prepared by a one-step and a two-step method. Glutaraldehyde was used as a cross-linking agent. Two methods were used for removing unconjugated HRP: Sephadex G-200 gel chromatography and ammonium sulfate precipitation. The conjugates were characterized immunologically, immunochemically and enzymatically. The immunohistoenzymic properties of the conjugates were tested on unfixed cryostat sections of the skin of patients with chronic discoid lupus erythematosus. The influence of the presence of unconjugated HRP and unconjugated IgG was studied. Optimal results were obtained with conjugates prepared by a two-step method. Removing unconjugated HRPimproved the immunohistoenzymic properties of the conjugates. Conjugated and unconjugated IgG could be separated by Sephadex G-200 gel chromatography.
Vacca, L L; Rosario, S L; Zimmerman, E A; Tomashefsky, P; Po-Ying, N G; Hsu, K C
doi: 10.1177/23.3.1127219pmid: 1127219
Immunoperoxidase techniques are presented which can be used to localize horseradish peroxidase-tracer in paraffin-embedded tissues of the central nervous system. Compared to histochemical methods using frozen sections, these immunologic techniques allow the use of stored, serial paraffin sections, and appear more sensitive for the demonstration of intraneuronal horseradish peroxidase after retrograde transport. The immunoperoxidase bridge techniques from reaction products of high quality which can easily be seen in fine processes.
doi: 10.1177/23.3.236341pmid: 236341
The use of cinnamyl nitroblue tetrazolium chloride (DS-NBT) in dehydrogenase experiments (lactic dehydrogenase, succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) in cytochrome oxidase experiments indicated that mitochondrial oxidoreduction reactions from nicotinamide adenine dinucleotide to cytochrome oxidase are located on the inner mitochondrial membrane in the outer compartment and the intracristate spaces. These reactions behave according to the chemiosmotic hypothesis. The cochlear hair cell mitochondria are cytochemically indistinguishable from free liver mitochondria. The heterogeneous mitochondrial staining pattern is related to the osmolarity of the incubation media, solubility of the enzymes and pH of the medium, but not to the fixation method.
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