Journal of Histochemistry & Cytochemistry

Blair, O C; Roti Roti, J L

doi: 10.1177/28.6.6771322pmid: 6771322

Exponentially growing Chinese hamster ovary cells were pulse labeled with 3H-leucine, 3H-lysine, or 3H-tryptophan, fixed, and stained by either the acriflavine-Feulgen procedure or with fluorescein isothiocyanate (FITC). Protein content as determined by FITC fluorescence was representative of protein content determined biochemically by the method of Lowry. Utilizing a fluorescence-activated cell sorter, 3H-labeled cells were sorted according to their DNA or protein content and the incorporation of 3H-leucine, 3H-lysine, or 3H-tryptophan determined by liquid scintillation counting. The rates of 3H-leucine, 3H-lysine, and 3H-tryptophan incorporation increased with respect to increasing DNA content (G1, mid-S, G2+M). The rate of 3H-lysine incorporation increased continuously with increasing protein content, whereas the rates of 3H-leucine and 3H-tryptophan incorporation were constant initially with an increase in incorporation near mid-cycle followed by a slight decrease. Matrix algebra modeling of the increase in protein content suggests that 3H-lysine incorporation is consistent with a sigmoidal increase in protein content, however, 3H-leucine and 3H-tryptophan incorporation do not follow either the exponential, linear, or sigmoidal models. Matrix algebra simulation of the FITC protein distribution indicates that while the rate of protein accumulation is not linear, the exponential and sigmoidal models fit the experimental data equally well.

Bauer, K D; Dethlefsen, L A

doi: 10.1177/28.6.6156196pmid: 6156196

Total RNA content in Chinese hamster ovary and HeLa-S3 cells determined by ultraviolet spectroscopy is compared with the red fluorescence distribution of acridine orange-stained cells observed by flow cytometry. A correlation coefficient of 0.93 is obtained when these methods of estimating RNA content are compared after various RNAse treatments. These data suggest that acridine orange staining effectively quantitates total cellular RNA content when analyzed by flow cytometry, although DNA is also shown to contribute a low but significant background of red fluorescence.

Tandler, C J

doi: 10.1177/28.6.7391548pmid: 7391548

Tissue amino groups undergo dithiocarbamylation on treatment (12 hr, 22-25 degrees C) with a carbon disulfide:triethylamine:tetrahydrofuran (1:1:0.5, v/v) mixture. After washing with tetrahydrofuran:triethylamine (4:1, v/v) followed by tetrahydrofuran:pyridine (4:1,v/v) and ice-cold distilled water tissues were immersed into ice-cold 10% (w/v) lead acetate to yield yellowish lead dithiocarbamates. At 22-25 degrees C the derivatives obtained from primary amines decompose to lead sulfide. After washing with 5% lead acetate in 10% acetic acid followed by three changes of distilled water tissues are dehydrated and embedded in paraffin or Maraglas as usual. In proteins the reaction is given by the epsilon-amino group of lysine and alpha-terminal amino groups, while nucleic acids do not react. Results are shown in tetrahydrofuran-delipidized ethanol or formalin-fixed 1) plant root tips where brown-colored and electron-dense lead sulfide deposits are localized in condensed chromatin and chromosomes attributable to lysine-rich histones and 2) rat skeletal muscle where an interfibrillar component is visualized in a repeating pattern, possibly related to terminal regions of the sarcoplasmic reticulum.

Gossett, L A; Hecht, R M

doi: 10.1177/28.6.6993551pmid: 6993551

A simple squash technique was developed which permits the observation of individual nuclei during embryogenesis of Caenorhabditis elegans. The technique consists of placing several two-cell stage embryos on a subbed slide in a droplet of M-9 salt buffer and incubating them in a sealed humidity chamber at 16.4 degrees C for increasing time intervals. The embryos are then squashed, fixed, and stained with Hoechst 33258. Rate of cleavage at 25.0 degrees C is 1.8 times faster than that at 16.4 degrees C. This yields superimposable growth curves upon correction for temperature. An initial lag in the rate of nuclear cleavage is followed by a burst of cell proliferation, which continues and then slows before 550-580 cells are produced at 4 to 5 hr at 25 degrees C. The squash size increases with cell number and reaches a maximum at about the 400-cell stage when early morphogenesis begins. The second half of embryogenesis is characterized by histogenesis in which the cells are held more tightly together, individual nuclei become less distinct, and the squash size decreases to a minimum as a small worm is formed.

Sugai, N; Ito, S

doi: 10.1177/28.6.6156197pmid: 6156197

The ultrastructural localization of carbonic anhydrase activity in mouse gastric mucosal cells as revealed by the cobalt bicarbonate histochemical method of Hansson has been made. In addition the effects of fixatives used for ultrastructural studies have been evaluated for reduction of carbonic anhydrase activity; exogenous erythrocyte carbonic anhydrase has been localized in tissues; acetazolamide and potassium cyanate inhibition of activity demonstrated; and an improved method for the osmication of reacted tissues for electron microscopy has been developed. The results indicate that the glutaraldehyde, formaldehyde, picric acid fixative, which retains about 5% of the original carbonic anhydrase activity, is distinctly better for histochemical studies than formaldehyde fixation, which retains about 32% activity. Acetazolamide at 10(-5) M consistently inhibits histochemical reaction, as does 20 mM KCNO, in the incubation medium. Exogenous carbonic anhydrase is readily visualized by the histochemical technique. Electron microscopy of gastric mucosa reacted for carbonic anhydrase activity indicates the focal deposition of the cobalt sulfide reaction product in the cores of microvilli lining the intracellular canaliculi, in the basal and lateral cell folds of parietal cells, and in the microvilli as well as the cytoplasm between mucous granules in the surface mucous cells. In addition, some reaction product was found in the mitochondrial cristae and in some nuclei and intercellular spaces.

Muirhead, K A; Irons, R D; Bruns, R; Horan, P K

doi: 10.1177/28.6.7391549pmid: 7391549

Characterization of a rabbit model system for the study of cell cycle effects of myelotoxic agents in normal bone marrow is described. Cell cycle phase distributions are obtained by computer analysis of flow cytometric single cell DNA histograms. Comparison of marrow aspirates with marrow samples from sacrificed animals indicates that dilution of aspirates with peripheral blood is not significant. Aspiration of marrow from one bone does not affect the cell cycle distribution of unsampled bones. Hence, sequential aspirates of different bones in a single animal may be used as representative samples for further study of effects of myelotoxins on marrow proliferation and differentiation.

Budd, G C; Barnard, E A; Porter, C; Mattimoe, S A

doi: 10.1177/28.6.7391550pmid: 7391550

Fluorophosphate-reactive (FPR) sites in the adult male rat liver, tentatively identified as esterase active centers, were localized and measured using the combined techniques of quantitative electron microscope radioautography and morphometric analysis with the light and electron microscope. The FPR sites were measured in liver which had been prefixed by perfusion with 1.5% glutaraldehyde and reacted with 10(-4) M tritium-labeled diisopropyl fluorophosphate (3H-DFP). Under the experimental conditions 64-67% of the esterase activity in fresh liver was retained for reaction with the 3H-DFP, which is known to bind irreversibly to the active sites of certain esterases. In light and electron microscope radioautographs the developed silver grains were concentrated over the cytoplasm of hepatocytes. A low concentration occurred over erythrocytes. All other areas in the liver had a concentration of grains resembling the background concentration. Quantitative measurements of grain density in the electron microscope radioautographs revealed the highest density, after correcting for radiation spread, in cytoplasmic granules (mainly cytolysomes). The grain densities over the rough and smooth endoplasmic reticulum and associated structures were also equal to or above the average hepatocyte grain density. Due to the large fractional volume of endoplasmic reticulum per hepatocyte (58% of cell volume) and the fraction of the liver occupied by hepatocytes (79% of liver volume) the majority of FPR sites in the liver occurred in the rough and smooth endoplasmic reticulum and associated structures. The average numbers of FPR sites were calculated per micrometer3 of hepatocyte (5.0 x 10(5) sites/micrometer3) and per unit volume of each significantly labeled organelle. In addition, the number of FPR sites per hepatocyte (2.5 X 10(9) sites/cell), per cm3 liver (4.1 X 10(17) sites/cm3) and in the total liver of an average 100 g male rate (2.2 X 10(18) sites/total liver) were also calculated.

Yokoyama, M; Chang, J P; Moller, P C

doi: 10.1177/28.6.6248591pmid: 6248591

Concanavalin A (Con A) binding sites and their mobility were studied by peroxidase (Po) and ferritin labeling techniques in normal and SV40 transformed human fibroblasts. Binding sites were visualized either as osmium black of 3'3-diaminobenzidine (DAB) reactions or as ferritin particles. DAB reaction products were localized at the external surface of the plasma membrane and in some multivesicular bodies of fixed cells. The labeling was continuous in normal and SV40 transformed human fibroblasts. When living cells were treated with Con A-Po at 4 degrees C and incubated at 37 degrees C, both normal and transformed cells showed remarkable changes. The foci of membrane indentations (caps or patches) are formed on the cell surface. Many labeled internalized vacuoles and vesicles appeared within the cytoplasm and in close proximity to the Golgi region of all cell types. The cellular changes occurred more quickly in transformed cells than in normal cells. It is concluded that normal cells do cap under certain conditions and that the plasma membranes of transformed cells are more fluid than those of normal cells.

Cocchia, D; Michetti, F

doi: 10.1177/28.6.6156198pmid: 6156198

Antibodies against the loosely bound subnuclear protein fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the ultrastructural distribution of the antigenic determinants in rat cerebellum was studied using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. A localization restricted to the nucleus was observed. Both neuronal and neuroglial nuclei exhibited antigens, whereas nuclei of pericytes and endothelial cells did not. The immunoreaction product was homogeneously distributed in dispersed chromatin and was absent from condensed chromatin, suggesting that the antigens were confined to the active regions of the genoma. The outer nuclear membrane and the nucleolus appeared to be free of the antigens, while a perinucleolar ring of immunoreaction was detectable. Liver preparations showed a nuclear reaction markedly weaker than the one for brain nuclei. Adsorption of the serum with isolated liver nuclei nullified the reactivity in liver tissue, whereas a sharp reaction was still observed in the cerebellum, indicating the subcellular reaction under examination to contain antigens specifically concentrated in the nervous system or unique to the brain.

Orstavik, T B; Brandtzaeg, P; Nustad, K; Pierce, J V

doi: 10.1177/28.6.6156199pmid: 6156199

The localization of kallikrein in human exocrine organs was studied with a direct immunofluorescence method. In the submandibular and parotid salivary glands, kallikrein was found apically in the striated duct cells whereas it was absent from the main excretory ducts or present only as a weak luminal rim. Kallikrein was not found in the acinar cells or in cells of the intercalated ducts. In the pancreas, kallikrein-specific fluorescence was seen in the granular portion of the acinar cells, whereas the islets of Langerhans and ductal cells were unstained.