Journal of Histochemistry & Cytochemistry

NOLTE, J.; PETTE, D.

doi: 10.1177/20.8.567pmid: 4402953

A gel film technique was adopted for kinetic enzyme activity determination in single cells using cryostat sections by means of a microscope photometer. The main principle of the method is a comparative activity determination, based on bipositional recording of initial reaction kinetics in two preselected measuring fields in the same tissue section. Systematic model experiments were performed to prove the fulfillment of the following conditions: (a) validity of Beer's law; (b) optimal concentrations of substrates, cosubstrates and dye within the reaction film, including evidence that the reaction rate is not limited by insufficient diffusion of these compounds; (c) proportionality between local enzyme concentration or thickness of tissue sections and the recorded reaction rate; and (d) specificity of the method as demonstrated by studying reaction rates in the absence of the substrate as well as in the presence of substrate and a specific inhibitor. The validity of the method was also examined by comparing the levels of succinate dehydrogenase activity in various rat tissues, as measured microphotometrically, with the enzyme levels as determined in homogenates. Microphotometric assays for nicotinamide adenine dinucleotide phosphate isocitrate dehydrogenase, lactate dehydrogenase and succinate dehydrogenase are described. Using these techniques it was found that: the periportal and central areas of the hepatic lobule in rat contain the same level of lactate dehydrogenase activity; succinate dehydrogenase activity is 1.6 times higher in the periportal area as compared to the centrilobular area. In experimental thyrotoxicosis, the ratio of enzyme activities in the periportal and central areas was 1.1.

NOLTE, J.; PETTE, D.

doi: 10.1177/20.8.577pmid: 5044279

Comparative microphotometric determinations of succinate dehydrogenase, triosephosphate dehydrogenase and lactate dehydrogenase activities in single white, red and intermediate fibers in cross-sections of rat rectus femoris muscle and soleus muscle were performed. White fibers have the highest triosephosphate and lactate dehydrogenase activities and the lowest succinate dehydrogenase activity. Red fibers are highest in succinate dehydrogenase activity and relatively low in triosephosphate and lactate dehydrogenase activities. The intermediate fibers have medium succinate dehydrogenase activity and relatively low triosephosphate dehydrogenase activity. In the soleus muscle, the triosephosphate dehydrogenase activity of the red fibers exceeds that of the intermediate fibers. A parallel increase in succinate dehydrogenase is observed in the red fibers of musculus soleus and musculus rectus femoris after thyroid hormone administration. There is some evidence, however, that the red fibers of the two muscles investigated vary with regard to their absolute succinate dehydrogenase activities and also the triosephosphate dehydrogenase to succinate dehydrogenase activity ratio.

REITH, ALBRECHT; SCHÜLER, BARBARA

doi: 10.1177/20.8.583pmid: 4114592

The relationship between cytochrome oxidase activity assayed biochemically with 3,3'-diaminobenzidine (DAB) as substrate and the resulting electron microscopic staining reaction has been investigated. Mitochondria isolated from both fresh and perfusion-fixed (1% glutaraldehyde and phosphate buffer for 1-2 min) rat liver showed a significant increase in oxygen consumption, as measured by an oxygen electrode, after addition of DAB and were further stimulated by adding cytochrome c. 2,4-Dinitrophenol and antimycin had no effect on the oxidation of DAB; cyanide and azide completely inhibited DAB oxidation, resulting in the absence of any mitochondrial staining reaction. Already after incubation for only 2 min mitochondria exhibited electron-dense deposits on the surface of the inner mitochondrial membrane away from the matrix. The results indicate that DAB donates electrons at the cytochrome c level to the electron transfer system of the respiratory chain and demonstrate the direct relationship between the electron microscopic staining reaction and cytochrome oxidase activity.

MORIARTY, G. C.; HALMI, N. S.

doi: 10.1177/20.8.590pmid: 4114593

The technique involving use of unlabeled antibody and the peroxidase-antiperoxidase complex was used to identify the adrenocorticotropin (ACTH)-secreting cell in the anterior pituitary lobe of the rat and to localize ACTH in it electron microscopically in ultrathin sections. The ACTH cell is star-shaped, with processes extending around other cells, and contains secretory granules of a maximal diameter of 300 mµ arranged peripherally along the plasma membrane. Stain was observed on secretory granules, around them, in the Golgi complex and in rough endoplasmic reticulum. One day after adrenalectomy, the ACTH cell is degranulated and the staining intensity of its remaining granules and cytoplasm is decreased, suggesting release of ACTH stores. If cortisol is given 6 hr after adrenalectomy, 18 hr later the ACTH cells are well granulated and the granules stain more intensely than normal. In addition, staining around the granules and throughout the cytoplasm is more intense, suggesting that an early effect of cortisol is to block release of ACTH. Twenty-one days after adrenalectomy, the ACTH cells are greatly increased in numbers and have complex, tortuous processes filled with intensely stained secretory granules.

STRAUS, WERNER

doi: 10.1177/20.8.604pmid: 4114594

The sites of intradermally injected horseradish peroxidase (HRP), of its antibody and of HRP-anti-HRP antibody complexes were investigated in the dermis of rabbits showing strong skin reactions of delayed hypersensitivity against HRP. Comparison was made with the location of intradermally injected HRP and its antibody in the dermis of immunized rabbits showing slight degrees of hypersensitivity of a nondelayed type and with the location of intradermally injected HRP in the dermis of nonimmunized animals. In all skin reactions of delayed hypersensitivity, a considerable proportion of the injected antigen was associated with collagen fibers, the reaction product appearing as "dots," "rodlets" and "strands" in linear array on individual collagen fibers in the interior of the bundles or as strands coating the outermost surface of the bundles. The reaction for the antibody was positive at the same sites. It was suggested that receptors for antigen-antibody complexes may be associated with collagen fibers. Free granular deposits containing antigen-antibody complexes seemed to originate from the collagen-associated granules after their enlargement and release. The extracellular granules often adhered to the antibody-positive surface membranes of lymphocytes. Other granular deposits (antigen-antibody complexes) seemed to be phagocytosed by monocytes (macrophages) and fibroblasts. In areas of tissue injury in the upper dermis, many mononuclear cells showed the reaction for the antibody on their cell surface. Aggregations of such cells were often located close to disintegrating bundles of collagen fibers. It was suggested that the encounter of the antigen and the antibody (antigen-antibody complexes) on the surfaces of cells and of collagen may trigger the release of cytotoxic factors. A few lymphocytes in the dermis of hypersensitive rabbits contained the specific antibody on their cell surface before skin lesions were elicited.

BOER, G. J.

doi: 10.1177/20.8.621pmid: 4625527

Using the colorimetric micromethod of Mattenheimer and deBruin for the assay of carbonic anhydrase activity, a considerable loss of substrate (CO2 gas) from the incubation medium was measured during spontaneous hydration. Adding gaseous CO2 and coating the surface of the incubation medium with oil were employed in order to prevent this loss, which otherwise led to serious errors in the measurement of the enzyme activity. A mixture of 80% paraffin and 20% hexadecane as surface coating was satisfactory for reducing the CO2 exchange to negligible proportions. With this modified method, the carbonic anhydrase activity of frozen-dried samples of rat neurohypophysis was studied as a possible "marker" for pituicyte activity following osmotic stress. The enzyme activity increased about 130% after 6 days of water deprivation. Experiments with perfused tissue, however, indicated that this increase in enzyme activity was caused by an increase in the blood content of the neural lobe.

WESTE, SANDRA M.; PENINGTON, DAVID G.

doi: 10.1177/20.8.627pmid: 4114595

The use of fluorochromes in the Feulgen reaction for the quantitative measurement of deoxyribonucleic acid (DNA) has been explored previously but is rarely applied. The occurrence of nonspecific fluorescence due to compounds other than DNA and the fading of the fluorochrome on excitation can cause variation in the measurements of nuclear fluorescence. The method of Ruch (in Introduction to Quantitative Cytochemistry, Wied, G. L., ed., Academic Press, 1966, p. 281) using auramine O as the fluorochrome has been modified to achieve improved nuclear fluorescence with a more linear rate of fading. A simple Zeiss microscope photometer has been used. Problems of instrumentation and optics, including lens changes during measurements on cells of widely varying sizes such as megakaryocytes, have been analyzed and correction factors have been calculated. Microfluorometric measurements of nuclear DNA are of the same order of accuracy as those made by integrating microdensitometry, without the need in the latter technique to compress large cells to a uniform thickness. Microfluorometry has been applied with the new staining method to analyze the ploidy distribution of bone marrow megakaryocytes.

LEE, SIN HANG

doi: 10.1177/20.8.634pmid: 4339767

A method is introduced to isolate and purify parietal cells from the rabbit stomach which has been briefly perfused with 1% glutaraldehyde via the celiac artery. The fixed mucosa is initially digested with 1% trypsin and subsequently with 1% collagenase solution. The parietal cells are more resistant to the effects of these proteolytic enzymes than the other cell types in the gastric mucosa and can be recovered and purified by centrifugation in a discontinuous sucrose gradient. Glutaraldehyde fixation reduces the tendency of cell clumping and, therefore, facilitates isolation of the separated cells. The final yield from each adult rabbit stomach varies from 0.1 to 0.4 ml packed parietal cells of about 97% purity determined by nuclear counts. The nuclei and mitochondria of most of the isolated cells appear to be intact on electron micrographs, but the cell membrane is focally disrupted; the nuclear deoxyribonucleic acid (DNA) can be stained with Feulgen technique and extracted with perchloric acid for quantitative determination. Biochemical studies on incorporation of thymidine-3H into DNA of the parietal cells have been carried out in normal young adult rabbits and in rabbits under chronic histamine stimulation. The results show that there is no significant incorporation of thymidine-3H by mature parietal cells in any of the two groups of animals, although the whole mucosa DNA hydrolysates always exhibit a high specific activity.

STAPLE, P. H.

doi: 10.1177/20.8.644pmid: 4114596

doi: 10.1177/20.8.649pmid: N/A