bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of living normal and leukemic mouse lymphocytes by fluorescein diacetate.
Authors: Szöllösi J , Kertai P , Somogyi B , Damjanovich S
Published: April 2011

The esterases activity of normal and acute leukemic mouse lymphocytes and that of their homogenates was investigated using fluorescein diacetate (FDA) as a fluorogenic substrate. The activity proved to be the same for the two cell populations as well as for the homogenates prepared from them. In cell suspensions, having different osmolalities, the rate of FDA hydrolysis decreased significantly with the increasing osmolality only in the case of intact leukemic lymphocytes. changes in the membrane and cytoplasmic viscosity caused by increased or decreased environmental osmolality of cell suspensions occurred in the same direction and to the same extent for both normal and leukemic cells. Fluorescein, the fluorescent product of the hydrolysis, accumulates in leukemic lymphocytes, whereas it easily effluxes form normal lymphocytes. A flow microfluorimetry analysis of the cell population revealed that the fluorescein content of large leukemic lymphocytes was three times higher than that of small, normal ones. The observed differences specific for leukemic lymphocytes might be useful in detecting leukemic transformation in an early stage of acute lymphoid leukemia.
A band of alkaline phosphatase activity in the crypts of mouse duodenal epithelium.
Authors: Bjerknes M , Cheng H
Published: April 2011

Isolated mouse duodenal epithelium, in the form of structurally intact crypt-villus units, was used to study the distribution of alkaline phosphatase with histochemistry. The tissue was incubated on ice in the medium of Hugon and Borgers (J Histochem Cytochem 14:629, 1966), with constant stirring to ensure uniform reaction. Continuous activity was observed frm the crypt mouth to the villus tip. A single band of alkaline phosphatase activity, 2-03 cell in height, was observed in the mid-crypt region in about 80% of crypts studied. Control studies (no substrate control, no lead control, pH control, inhibitor control, inactivated enzyme control, no enzyme control, and stimulator indeed due to alkaline phosphatase. The narrow band of activity in the crypt was also observed in vibratome sections of nonfrozen tissue. When isolated epithelium was subjected to a freeze-thaw cycle and then incubated in Hugon's medium, reaction product was observed continuously from the mid-crypt region to the villus tip. This pattern was similar to that observed with frozen sections. We conclude that alkaline phosphatase is present in an active form in epithelial cells in the region of the band. In epithelial cells above the band, e.e., in the upper crypt, alkaline phosphatase is present in an inactive form which may be activated by a freeze-thaw cycle.
Selective inhibition of nonspecific eosinophil staining or identification of eosinophilic granulocytes by paired counterstaining in immunofluorescence studies.
Authors: Valnes K , Brandtzaeg P
Published: April 2011

Nonspecific staining of eosinophilic granulocytes produced by nonimmune rabbit IgG conjugates, highly labeled with fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (MRITC), was efficiently inhibited by pretreatment of tissue sections with diaminobenzidine and hydrogen peroxide; the brown reaction product developed in the presence of this substrate by the endogenous peroxidase of the granulocytes sheltered these cells from subsequent nonspecific staining without interfering with specific immunofluorescence staining of other tissue elements. Similar results were obtained with the alternative noncarcinogenic peroxidase substrate, p-phenylenediamine-pyrocatechol. Alternatively, the eosinophils could be identified by their nonspecific staining with FITC-labeled bovine serum albumin when this conjugate was combined with an MRITC conjugate used for specific staining; optical conditions selective for green and red fluorescence discriminated distinctly between double-stained eosinophils and purely red specifically stained cells.
Immunohistochemical localization of indoleamine 2,3-dioxygenase in the argyrophilic cells of rabbit duodenum and thyroid gland.
Authors: Watanabe Y , Yoshida R , Sono M , Hayaishi O
Published: April 2011

To identify the cells containing indoleamine 2,3-dioxygenase, indirect immunofluorescence studies were performed using the monospecific antibody toward the enzyme purified to apparent homogeneity from rabbit small intestine. The antibody precipitated almost 100% of the enzyme activities of various organs or rabbit in a quantitatively similar manner, but did not cross-react with tryptophan 2,3-dioxygenase [L-tryptophan:oxygen 2,3-oxidoreductase (decyclizing), E.C. 1.13.11.11] in the liver. Immunohistochemical localization was studied using rabbit tissue because of the species-specific reactivity of the antibody. In the duodenum, indoleamine 2,3-dioxygenase-immunoreactive cels were found to be sporadically but exclusively localized in the mucosa, a large number being in the basal portion. No staining was seen in the consecutive sections treated with control immunoglobulin. Restaining within the same section using Grimelius' silver technique revealed that the immunoreactive cells coincided with the argyrophilic cells reportedly containing serotonin under physiological conditions. In the thyroid gland, the immunofluorescence was positive in the cluster of the parafollicular cells also shown to be argyrophilic, the cells proposed to have a monoamine-handling capacity specific for serotonin. These results indicate that indoleamine 2,3-dioxygenase is present in those cells rich in serotonin or active in serotonin biosynthesis and further support our previous contention that the enzyme may play an important role in the regulation of the metabolism of indoleamines and their precursors in such cells.
OTO method for preservation of actin filaments in electron microscopy.
Authors: Aoki M , Tavassoli M
Published: April 2011

Osmium tetroxide, commonly used as a fixative in electron microscopy, can destroy actin filaments. Thiocarbohydrizide (TCH) is a bipolar substance that binds to the osmium. By sandwiching TCH between two phases of osmium treatment, tissue exposure to osmium could be minimized without destroying actin filaments. The contrast of osmophilic components of cells was also enhanced.
Immunocytochemical analysis of the rat pineal gland using antisera generated against luteinizing hormone-releasing hormone (LHRH).
Authors: Piekut D T , Knigge K M
Published: April 2011

Seven different antisera generated against synthetic luteinizing hormone-releasing hormone (LHRH) were used to stain pineal gland and median eminence of the hypothalamus of normal rats. The LHRH immunogen complexes used to generate these antisera produced either N- or C-terminal directed antibodies or antisera that recognized both portions of the hormone molecule. Of these, four revealed LHRH-like immunoreactive material in the median eminence and pineal gland, two stained median eminence and not pineal, and one was positive in pineal only. These observations suggest that immunoreactive material in pineal gland may be an LHRH-like substance whose affinity characteristics are different from that of hypothalamic LHRH.
Fluorescence demonstration of dipeptidyl peptidase II in skeletal, cardiac, and vascular smooth muscles.
Authors: Stauber W T , Ong S H
Published: April 2011

Dipeptidyl peptidase II (Dpp II) was demonstrated histochemically in soleus, extensor digitorum longus, cardiac, and vascular smooth muscle tissues using Lys-Ala-4-methoxy-beta-naphthylamide or Lys-Pro-4-methoxy-beta-naphthylamide as the substrate. The enzyme was found to be localized in discrete granules in all muscle types, but varied in its apparent activity. Dpp II activity was greatest in cardiac and least in extensor digitorum longus muscles with activity in soleus and vascular smooth muscles in between these extremes. Since Dpp II is localized only in lysosomes, the relative amounts and locales of lysosomes can be easily observed in normal muscle cells by the techniques described in this study.
An electron microscopic histochemical and analytical X-ray microprobe study of calcification in Bruch's membrane from human eyes.
Authors: Davis W L , Jones R G , Hagler H K
Published: April 2011

Transmission electron microscopy, electron microprobe analysis, high temperature microincineration, and electron microscopic histochemical procedures were used to study the electron-dense deposits characteristic of the macular aspect of aged human eyes. These inorganic deposits were rich in calcium and phosphorus and selectively removed by flotation on formic acid. The amorphous decalcified masses showed a significant sulfur peak and were readily stained with acidic phosphotungstic acid. The latter observations are indicative of the presence of organic matrical proteoglycan. Such data may be a further indication that proteoglycans are retained at sites of calcification.
Effect of actinomycin-D on the staining of whole cells with acridine orange.
Authors: Chambers S H , Watson J V
Published: April 2011

Flow cytofluorometric techniques have been used to investigate the interference of actinomycin-D with the staining of acridine orange in whole cells. The results show that reduction in fluorescence intensity will only occur at actinomycin-D concentrations greater than 10(3) ng/ml in our system, providing the drug is washed out prior to staining.
Improved contrast in cytochemistry of dehydrogenases by scanning transmission electron microscopy.
Authors: Deimann W , Freeman R , Fahimi H D
Published: April 2011

A scanning transmission electron microscope (STEM) was used to examine ultrathin sections of rabbit white skeletal muscle. Lactic dehydrogenase (LDH) activity was localized in the tissue using the tetra-nitro blue tetrazolium (TNBT) method. For most specimens postfixation was omitted in order to avoid reoxidation and solubilization of the formazan by osmium tetroxide. The STEM image revealed sufficient contrast of the intracellular structures and apparently electron-dense reaction product in the sarcoplasmic reticulum and mitochondria. Substantially less contrast was obtained when the same areas were observed by conventional transmission electron microscopy (CTEM). In material postfixed with osmium tetroxide, although the tissue contrast was improved, the TNBT reaction product was focally leached out, exhibiting lower contrast than in unosmicated sections. These results indicate that the fine structural visualization of dehydrogenases with TNBT, the STEM technique as used in the present study is superior to that obtained by CTEM.
Heparan sulfate of AH-130 ascites hepatoma cells: a cell-surface glycosaminoglycan not displaced by heparin.
Authors: Hurst R E , Parmley R T , Nakamura N , West S S , Denys F R
Published: April 2011

This study reports on the ultrastructural location and biophysical properties of cell-associated glycosaminoglycans of AH-130 cells, an azo dye-induced ascites hepatoma. Earlier studies have shown that a low-sulfated heparan sulfate, which comprises 93% of their total glycosaminoglycan (GAG) content, is associated with these cells. High-iron diamine, an ultrastructural stain for sulfated glycoconjugates, stained the hepatoma cell surfaces heavily. With the exception of occasional light staining in a few cytoplasmic granules, intracellular organelles did not stain with this method. The lack of an extensive pool of intracellular GAG was confirmed by quantitative fluorescence microscopy of cells vitally stained with acridine orange. The nature of the binding of the cell-surface heparan sulfate was explored by competitive binding studies with exogenous heparin. When cells were incubated with exogenous heparin, release of heparan sulfate into the medium was not detected, although heparin was bound. We conclude that low-sulfated heparan sulfate is an integral component of the AH-130 hepatoma cell surface and is bound at a site different than heparin.
Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin.
Authors: Takagi M , Parmley R T , Denys F R
Published: April 2011

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicer's hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiéry's periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.
Cytochemical localization of ornithine decarboxylase with rhodamine or biotin-labeled alpha-difluoromethylornithine. An example for the use of labeled irreversible enzyme inhibitors as cytochemical markers.
Authors: Gilad G M , Gilad V H
Published: April 2011

The present work describes a new method for cytochemical localization of enzymes using ornithine decarboxylase (ODC) as an example. The method is based on the preservation of the characteristic-specific and irreversible binding of the inhibitor alpha-difluoromethylornithine (alpha-DFMO) following its conjugation to "label" molecules. The inhibitor has been conjugated to the fluorescent molecule rhodamine-B-isothiocyanate, and its localization in tissue sections was detected directly by fluorescence cytochemistry. Alternatively, alpha-DFMO has been conjugated to biotin and its cytochemical localization determined indirectly following its binding with avidin conjugated to horseradish peroxidase (HRP) and visualization of the HRP reaction product. Both labeled inhibitor molecules were successfully localized cytochemically within specific cells of the developing rat cerebellum and rat liver following thioacetamide injection where ODC activity is greatly enhanced. This novel technique should be of general application 1) in other tissues, 2) for other enzymes, and 3) in electron microscopic studies for ultrastructural localization of the enzyme.
Immunocytochemical detection of peptides in osmicated and plastic-embedded tissue. An electron microscopic study.
Authors: Pelletier G , Puviani R , Bosler O , Descarries L
Published: April 2011

In an attempt to improve the ultrastructural preservation of tissue immunostained with the postembedding staining technique, we verified the effect of postfixation with OsO4 on the antigenicity of several pituitary hormones and neuropeptides. For this purpose, the rat pituitary and central nervous system (CNS) were perfused with 2.5% glutaraldehyde and postfixed in two different ways: a) postfixation by immersion of small fragments in a mixture of 1% OsO4 and 1% potassium ferrocyanide, and b) postfixation with perfusion of 500 ml of 0.5% OsO4. The results obtained were similar with the two types of postfixation. In the pituitary gland, all the hormones could be very easily detected, although the staining was less intense for the glycoprotidic hormones. In the CNS the following neuropeptides: somatostatin, luteinizing hormone-releasing hormone (LHRH), Leu-enkephalin and substance P could be immunostained. These results indicate that a variety of polypeptidic antigens, including small neuropeptides, can survive postfixation with OsO4. In both the pituitary and CNS, the ultrastructural preservation was very good with a high contrast of membranes, thus permitting a clear identification of positive organelles. Moreover, it appears that, since the synaptic junctions are well-preserved after postfixation with OsO4, postembedding staining can now be utilized to identify and classify the different categories of endings containing neuropeptides.
Simple adaptations to extend the range of flow cytometry five orders of magnitude for the DNA analysis of uni-and multicellular systems.
Authors: Hecht R M , Schomer D F , Oró J A , Bartel A H , Hungerford E V
Published: April 2011

Procedures and instrumentation are described to extend the capability of a cytometry system to record samples that exhibit a wide range of fluorescence such as multicellular systems. The method employs a log amplifier in combination with a set of neutral density filters that reduces the incident light reaching the photomultiplier tube. With any given filter, signals within an intensity range of 200-fold can be measured; different filters can be used to obtain an extended overall range. Polystyrene fluorescent microspheres and a variety of mithramycin stained biological samples ranging from yeast cells to Paramecium were processed by the system. The relative DNA content of individual multicellular embryos was determined for a heterogeneous population of embryonic stages isolated from the nematode, Caenorhabditis elegans. As part of the evaluation of the procedure, the practical upper limit of range extension was determined. The most intense fluorescent signal was produced when untreated pecan pollen stained with ethidium bromide fluoresced with a factor (8.4 +/- 1.3) X 10(4) more than ethidium bromide stained E. coli cells.
The use of monoclonal antibodies against human chorionic gonadotropin for immunoperoxidase staining of normal placenta, pituitary gland, and pituitary adenomas.
Authors: Wahlström T , Stenman U H , Lundqvist C , Tanner P , Schröder J , Seppälä M
Published: April 2011

A monoclonal mouse antibody to human chorionic gonadotropin (hCG) was used in a modified unlabeled antibody enzyme-bridge staining method to demonstrate the localization of hCG in normal human placenta, pituitary gland, and six pituitary chromophobe adenomas. Mouse ascitic fluid containing monoclonal antibody could be diluted up to 1:500,000 for detection of hCG in the syncytiotrophoblast, whereas no staining was observed in the pituitary or adenomas even with high antibody concentrations (dilutions from 1:500 upward). Nonspecific background staining was negligible. These results demonstrate that monoclonal antibodies are suitable for immunohistochemical localization of antigens in tissues.
Immunocytochemical localization of receptor human chorionic gonadotropin complexes in rat leydig cells.
Authors: Rajaniemi H , Karjalainen M , Veijola M , Ritanen-Kaivamo S , Kellokumpu S , Metsikkö K
Published: April 2011

Localization of receptor-bound human chorionic gonadotropin (hCG) in rat testis was studied by the peroxidase-antiperoxidase (PAP) complex method. The rats were injected with a single intravenous dose (1000 IU) of hCG. Three, 6, 12, and 24 hr after injection the testes were removed for localization of the hormone. The hormone localized to the periphery of the Leydig cells at all observation points. The intensity of the staining varied between the cells, suggesting that the number of receptors or the accessibility of the receptors to the circulating hormone varies from one cell to another. The staining surrounded the Leydig cells unevenly, but no progressive patching or capping was found. This observation suggests that hCG binds preferentially to the cell surface areas directed toward the capillaries. Compatible results were obtained with anti-hCG serum and with antisera against the hCG subunits. These results are consistent with previous observations that the luteinizing hormone (hCG) receptors accessible to the circulating hormone are located at the surface of the Leydig cells.
Correlated biochemical and cytochemical studies of nicotinamide adenine dinucleotide phosphatase (NADPase) activity in ameloblasts using structural analogues of NADP.
Authors: Smith C E
Published: April 2011

The effects of altering the molecular structure of nicotinamide adenine dinucleotide phosphate (NADP) on enzymatic hydrolysis of the monoester phosphate group was examined at pH 5.0 in rat incisor ameloblasts using eight analogues of the oxidized form of the beta-isomer of NADP (beta-NADP+) modified in either the nicotinamide or adenine regions, or at the site for attachment of the monoester phosphate group to the molecule. Biochemical studies with whole homogenates of unfixed enamel organs revealed that the Michaelis-Menten constant (KM) and the maximum rate of dephosphorylation (Vmax) were different for these analogues relative to values estimated with beta-NADP+ as substrate. For example, the KM value was 3-fold higher and the Vmax value was lower by about 1/2 with the reduced form of the molecule as substrate. The KM value was about 2-fold higher but the Vmax value was about the same with an analogue lacking the nicotinamide group as substrate, while both the KM and the Vmax values were about 2-fold higher with an analogue containing an ethylene group covalently linked to adenine as substrate. In contrast, the KM value was markedly reduced (1/15) and the Vmax value was elevated (4-fold) using an analogue as substrate which contained the phosphate group attached at the 3'-position (3'-NADP+), rather than the 2'-position as in beta-NADP+ and other analogues. Cytochemical studies with glutaraldehyde-fixed enamel organs revealed that reaction product from hydrolysis of beta-NADP+ and other analogues containing the phosphate group attached at the natural 2'-position was localized within the intermediate saccules of the ameloblast Golgi apparatus. Reaction product from hydrolysis of 3'-NADP+, however, was localized at a different site, that is, within granules and membranous connections of the GERL system in the cell. Hence, 3'-NADP+ was not hydrolyzed by NADPase but by another enzyme tentatively identified as Coenzyme A phosphatase.
Immunocytochemical localization of catalase in rat liver.
Authors: Yokota S , Fahimi H D
Published: April 2011

The intracellular localization of catalase has been studied using monospecific Fab fragments against rat liver catalase (RLC) and preembedding immunoelectron microscopy. Purified RLC, exhibiting a single band on sodium dodecyl sulfate gel electrophoresis, was used for the immunization of rabbits. The anti-RLC IgG was purified by affinity chromatography. Fab fragments were obtained by papain digestion and were labeled with horseradish peroxidase (HRP) using a modified two-step procedure and glutaraldehyde as coupling agents. Livers were perfused with 4% depolymerized paraformaldehyde and chopper sections were incubated with HRP-labeled Fab fragments against RLC. Because of the limited penetration of labeled Fab fragments into chopper sections a simple method for preparation of a cell suspension from aldehyde-fixed livers was devised. Adequate staining of more than 90% of cell was obtained by incubation of cell suspensions for 12--18 hr with the labeled antibody. By light microscopy specific staining was present in fine granules in the cytoplasm of hepatocytes. By electron microscopy the electron-dense reaction product was localized in the matrix of peroxisomes with no reaction in the endoplasmic reticulum and the Golgi complex. In some hepatocytes, positively reacted peroxisomes were seen side by side with unstained particles. Although focal diffusion was noted around a few peroxisomes, no evidence of cytoplasmic catalase independent of peroxisomes was found. These observations indicate that in rat liver peroxisomes are the only organelle containing substantial amounts of catalase antigen and rule out any involvement of the endoplasmic reticulum and the Golgi complex in the sequestration of this protein.
A rapid method for immunofluorescent staining of paraffin sections using iron-containing protein A microspheres.
Authors: Widder K J , Senyei A E , Burchette J L , Ovadia H , Paterson P Y
Published: April 2011

A method to rapidly perform immunofluorescence or light microscopic staining on formalin-fixed paraffin sections has been devised utilizing magnetic albumin microspheres containing Staphylococcal protein A. Because the protein A constituent of the microspheres has the property of binding the Fc portion of immunoglobulin G (IgG) class antibodies, the microspheres can be used to rapidly bind antigen-antibody complexes by the Fc portion of the antibody. Deparaffinized sections were stained with fluorescein isothiocyanate-conjugated antibody (IgG fractions) by standard techniques, after which the protein A microspheres were layered over the sections. Distinct fluorescence of sections was noted with the addition of the microspheres, whereas only autofluorescence was present with direct staining alone. The microspheres were also visualized by light microscopy by a subsequent Prussian blue reaction, staining the Fe3O4 within the microsphere matrix. This method represents a more rapid method for identifying antigens in tissues embedded in paraffin than has previously been reported.
Retrospective demonstration of endogenous peroxidase activity in plastic-embedded tissues conventionally prepared for electron microscopy.
Authors: Del Cerro M , Cogen J P , Del Cerro C
Published: April 2011

A procedure is described that permits retrospective demonstration of intracellular endogenous peroxidase activity in tissue conventionally prepared for electron microscopy, i.e., doubly fixed with aldehydes and osmium tetroxide, "stained" in block with uranyl acetate, and embedded in epoxy resins. Using sodium ethoxide, plastic was removed from 1 micrometer sections; subsequently, the sections were incubated for 20 min in diaminobenzidine solution (44 mg/100 ml) made in acetate-citric acid buffer, pH 5.6, with 0.01% hydrogen peroxide. After this treatment, the sections were rinsed, dehydrated, and mounted. Cell types known to have endogenous peroxidase activity (red blood cells, macrophages, and retinal pigment epithelium cells in our preparations) show positive granules in their cytoplasm--control sections were uniformly negative. This method, which could also be used prospectively, cytochemically demonstrates endogenous peroxidase activity upon optical microscopical examination of the treated tissues; correlative electron microscopic studies may be performed on the same tissue block, or even adjacent sections.
Inhibition of endogenous tissue alkaline phosphatase with the use of alkaline phosphatase conjugates in immunohistochemistry.
Authors: Ponder B A , Wilkinson M M
Published: April 2011

In mammals there are two forms of alkaline phosphatase, one of which is widely distributed in a variety of tissues, and one of which is confined to intestine. Levamisole (1-tetramisole) inhibits the nonintestinal form of the enzyme, but is without effect on the intestinal form. We have exploited this difference by using conjugates made with calf intestinal alkaline phosphatase for immunohistochemical demonstration of H2 antigens in frozen section of mouse tissues. The alkaline phosphatase staining is performed in the presence of 1 mm levamisole, which inhibits the endogenous tissue enzyme without loss of staining by the conjugate. Endogenous enzyme can be inhibited by other means, such as exposure to 20% acetic acid, but labile antigens may be destroyed.
Freeze-fracture cytochemistry: thin sections of cells and tissues after labeling of fractures faces.
Authors: da Silva P P , Parkison C , Dwyer N
Published: April 2011

Experimental details of a new method for the cytochemical characterization of the membrane faces and cytoplasm produced by freeze-fracture of isolated cells and tissues are presented. This new method-"fracture-label"-involves grinding of frozen samples immersed in liquid nitrogen, thawing, cytochemical labeling of the fractured faces, and processing for thin section electron microscopy. Cationized ferritin (at pH. 7.5 and 4.0), colloidal iron, as well as concanavalin A are used to label the fractures faces of leukocytes and Hela cells embedded in a cross-linked matrix of bovine serum albumin and of liver and spleen tissues. Our results show the presence of numerous anionic binding sites on the fracture faces of all plasma and cytoplasmic membranes, and of concanavalin A binding sites preferentially associated to the exoplasmic fracture faces of plasma and nuclear envelope membranes. A proportion of the anionic sites appears to be revealed by, or during, the freeze-fracture process. Colloidal iron labeling also shows preferential association with the chromatin areas of cross-fractured nuclei. The results show that "fracture-label", i.e., the combined application of freeze-fracture and cytochemical labeling techniques, can be used to study the surface chemistry of the fractures faces of biological membranes as well as of cross-fractured cytoplasm.
The relationships between the Golgi apparatus, GERL, and lysosomes of fetal rat liver Kupffer cells examined by ultrastructural phosphatase cytochemistry.
Authors: Pino R M , Pino L C , Bankston P W
Published: April 2011

Kupffer cells are the sinusoidal macrophages of the liver. Using ultrastructural phosphatase cytochemical methods, we examined the relationship between the Golgi apparatus, GERL, and lysosomes of Kupffer cells in fetal rat livers identified, in part, by their ability to phagocytize intravenously injected latex spheres. Thiamine pyrophosphatase (TPPase) activity was localized to the inner Golgi saccules and some vesicles in the Golgi region but not to GERL. A TPPase-like activity, demonstrable in lysosomes, was abolished by sodium fluoride but not suppressed by the alkaline phosphatase inhibitors L-cysteine and L-p-bromotetramisole. Acid phosphatase (AcPase) was localized by GERL, some coated vesicles, and in lysosomes, but not to the Golgi stacks. Continuities between GERL and lysosomes were observed. Phagosomes containing internalized latex spheres received TPPase and AcPase sequentially. TPPase was localized in phagosomes immediately after latex administration. AcPase activity was not found here until at least 10 minutes following the injection of the particulates. Our findings indicate that Kupffer cell lysosomes are derived from GERL, but also suggest that phagosomes may receive material packaged by the Golgi apparatus as well as GERL.
Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes.
Authors: Willingham M C , Rutherford A V , Gallo M G , Wehland J , Dickson R B , Schlegel R , Pastan I H
Published: April 2011

Concentrative receptor-mediated endocytosis of many specific ligands by cultured fibroblasts occurs through the coated pit-receptosome pathway. The formation of receptosomes was studied using two impermeant electron-dense labels for the cell surface, ruthenium red and concanavalin A-horseradish peroxidase. These studies show that at 4 degrees C, virtually all coated structures near the plasma membrane are in communication with the cell surface, and are not isolated coated vesicles. On warming cells to 37 degrees C for only 1 minute, a major portion of these structures become cryptic, that is, not labeled by these surface markers. However, on cooling cells immediately back to 4 degrees C, virtually all of these structures are again in communication with the surface. Many images showed that membrane of these cryptic pits to be continuous with the cell surface when caught in the appropriate plane of section; often there was a very narrow entrance that excluded extracellular label. At 37 degrees C, receptosomes could be occasionally seen forming as an invagination of membrane adjacent to the coated region. Mechanisms by which receptosomes may form and other evidence demonstrating the failure of coated pits to pinch off to form isolated coated vesicles during endocytosis are discussed.
Stereological methods in cell biology: where are we--where are we going?
Authors: Weibel E R
Published: April 2011

The current state of the art in morphometric cell biology is reviewed by looking at the developmental state of stereological methods, and at the approaches used to arrive at quantitative structure-function correlation. Stereological methods have reached a fairly advanced level of sophistication since mathematical stereology has been developed as a branch of geometric probability theory. The application of these methods in cell biology lags behind, both quantitatively and qualitatively. Among the strategies used in exploiting stereological methods in cell biology the physiological approach (where a change is induced experimentally and its effect on the cells is followed by biochemical and morphometric methods) ranks highest and is still valid. More analytical approaches, such as combining stereology and biochemistry in cell fraction studies, are fraught with difficulties. In considering future developments of stereological methods, the emphasis will have to be 1) on developing procedures for eliminating biases such as section thickness or resolution effects, and 2) on increasing the efficiency of the methods by better sampling rules and improved instrumentation. The future trends in morphometric cell biology might best be served by exploiting the potentials of histochemistry and stereology by combining them with a view to 1) establishing procedures for cell-specific sampling and 2) developing methods towards "molecular morphometry" on the basis of immunocytochemical labeling.
Immunoperoxidase localization of secretory component in reactive mesothelium and mesotheliomas.
Authors: Ernst C S , Brooks J J
Published: April 2011

Immunoperoxidase localization of the secretory component (SC) of immunoglobulin (Ig) A was undertaken on paraffin-embedded tissue to investigate its usefulness in differentiating epithelial from mesothelial neoplasms. Epithelial cells, but not mesothelial cells, have been previously reported to contain SC, which may be involved in the intraepithelial transport of secretory IgA. In this study we report, for the first time, focal but unequivocal staining for SC in reactive mesothelium and mesotheliomas (10/16). Reactivity was not associated with any histologic subtype of malignant mesothelioma although staining was usually seen in the "epithelial" type. The significance in terms of possible mesothelial secretory IgA transport is discussed.
A CHEMICALLY SPECIFIC DOUBLE-STAINING TECHNIQUE FOR DESOXYRIBOSENUCLEIC ACID AND OTHER ALDEHYDE-YIELDING OR ALDEHYDE-CONTAINING SUBSTANCES
Authors: DELAMATER EDWARD D. , SCHAECHTER MOSELIO , HUNTER M. ELIZABETH
Published: April 2011

A chemically specific double staining procedure has been devised for Chlamydomonas, which depends upon the staining of the phosphoric acid moiety of the chromosomal DNA in hydrochloric acid hydrolyzed cells, with the subsequent transfer of the basic dye (azure A) to the aldehyde radicals of the DNA in the presence of sulfurous acid. Subsequent treatment with Schiff reagent stains the primary zygote membrane magenta by combining with its free aldehyde groups. The method as outlined is simple and useful with potentials for application to other types of cells.
AN EVALUATION OF HISTOCHEMICAL METHODS FOR PROTEIN GROUPS
Authors: BURSTONE M. S.
Published: April 2011

The dinitrofluorobenzene, the ninhydrin-Schiff, alloxan-Schiff, and chloramine-Schiff methods are useful for the demonstration of certain protein groups in tissues.

The coupled tetrazonium and nitrobenzoylation methods present certain difficulties in interpretation. However, the latter may be of value as a collagen or elastic tissue stain.
FLAME PHOTOMETRIC DETERMINATION OF POTASSIUM IN MICROGRAM QUANTITIES OF TISSUE AND THE DISTRIBUTION OF POTASSIUM AND LIPID IN THE ADRENAL OF THE MONKEY AND GUINEA PIG (STUDIES IN HISTOCHEMISTRY XXXII)
Authors: GLICK DAVID , SWIGART RICHARD H. , NAYYAR SOM N. , STECKLEIN HELEN R.
Published: April 2011

A flame photometric method for the determination of potassium in microgram quantities of tissue was described.

A simple water extraction of the potassium from microtome sections of adrenal tissue was shown to be preferable to either digestion or dry-ashing in the preparation of the sample for analysis.

The quantitative histological distributions of total dry matter, total lipid, and potassium in the adrenal glands of M. rhesus and cynomolgus monkeys and guinea pigs were determined. No significant strain or species differences were observed among the data collected.

The total lipid in the fascicular zone reached maximum concentrations of 55-70% of the total dry-weight. The potassium concentration remained within the approximate range of 1.4-1.7% of the fat-free dry-weight throughout all of the regions of the adrenals of the animals used.
THE DISTRIBUTION OF PHOSPHOMONOESTERASES AND PYROPHOSPHATASE IN THE PARTICULATE FRACTIONS OF DOG CEREBRUM
Authors: WAKED NABIL , KERR STANLEY E.
Published: April 2011

Dog cerebrum was homogenized in 0.25M sucrose and fractionated by differential centrifugation to yield a "nuclear" sediment, three cytoplasmic particulate fractions and the final supernate. Each of these fractions was found to contain acid and alkaline phosphatase, 5'-nucleotidase, and pyrophosphatase activity. With the exception of alkaline phosphatase, the nuclear fraction contains very little enzyme activity, which may be due to cytoplasmic contaminants. The high alkaline phosphatase activity of the nuclear fraction originates from components other than the cell nuclei. The cytoplasmic particles contain a low percentage of the total alkaline phosphatase and pyrophosphatase activities, but relatively more of the acid phosphatase and 5' nucleotidase. The final supernate contains almost all of the pyrophosphatase activity. The specific activity of the three phosphomonoesterases in the supernate is low. The localization of the Nissl substance in the cytoplasmic particulate fractions is discussed.
HISTOCHEMICAL DETECTION OF PHOSPHORYLASE IN ANIMAL TISSUES
Authors: TAKEUCHI TADAO , KURIAKI HIDEO
Published: April 2011

Amylophosphorylase in animal tissues has been demonstrated in frozen sections by use of activators and primer in a modified Yin and Sun method. A suitable technique for this purpose was newly devised. It was experimentally proved that the synthesis of glycogen from glucose-1-phosphate in tissues was specifically due to phosphorylase activity itself. Amylophosphorylase was histochemically demonstrated in muscle fibers of skeletal and heart muscles, smooth muscles in certain organs, epithelium of esophagus etc. The reactions were shown in cytoplasms and the nuclei were not stained. The phosphorylase reaction of muscle was very closely related to striated structure in muscle fibers.
THE SPECIFICITY OF THE HISTOCHEMICAL METHOD FOR ADENOSINE TRIPHOSPHATAS
Authors: PADYKULA HELEN A. , HERMAN EDITH
Published: April 2011

Three categories of phosphatase activity toward adenosine triphosphate at pH 9.4 have been isolated histochemically: (1) phosphatase which requires -SH groups; (2) phosphatase which is inhibited by -SH groups; and (3) phosphatase which is relatively indifferent to -SH groups. This enzymatic activity was demonstrated in thin (5 µ), unfixed, frozen sections.

True adenosine triphosphatase has been separated histochemically by virtue of its -SH dependence. It was demonstrated in cardiac and skeletal muscle fibers and in the mitochondria of the kidney tubules. The staining of these structures can be prevented by treatment with an -SH inhibitor, such as p-chloromercuribenzoate or salyrganic acid. Also their stainability can be restored by reversing the inhibition of the enzymes with -SH compounds, such as BAL or cysteine. There was no reaction of these particular structures when adenosine diphosphate or adenosine-5-phosphate was used, suggesting that under the conditions of this histochemical test only the terminal phosphate of adenosine triphosphate is removed. Phosphatase activity of the smooth muscle of the intestinal muscularis toward adenosine triphosphate differed from that of striated muscle in that it was less sensitive to p-chloromercuribenzoate.

Sulfhydryl compounds, such as BAL or cysteine, enhanced the staining of the above mentioned sites of true adenosine triphosphatase activity. On the other hand, these -SH compounds are powerful inhibitors of alkaline phosphatase activity, especially toward adenosine-5-phosphate. When adenosine triphosphate was the substrate, BAL prevented the staining of the brush borders of the kidney tubules and intestinal epithelium, thus suggesting the participation of alkaline phosphatase in the dephosphorylation of adenosine triphosphate. Furthermore, the staining of these brush borders was unaffected by the -SH inhibitors.

The strong phosphatase activity of endothelium and vascular smooth muscle toward adenosine triphosphate was seemingly indifferent to -SH groups, since the staining of these structures was not markedly influenced by -SH inhibitors or compounds. The nature of this staining has not been elucidated.

Comparisons of the localization of phosphatase activity toward adenosine triphosphate, -diphosphate or -5-phosphate were made in the ventricle, tongue, kidney, and duodenum. Differences in the localization of phosphatase activity toward adenosine triphosphate and adenosine-5-phosphate were also made in liver, lung, spleen, uterus, and aorta.
SIGNIFICANCE OF COUPLING RATE IN HISTOCHEMICAL AZO DYE METHODS FOR ENZYMES
Authors: DEFENDI V. , PEARSE A. G. E.
Published: April 2011

1) In order to estimate the significance of the coupling rate in histochemical localization of enzymes a series of naphthols has been tested, using a streaming method with photoelectric recording. The naphthols were those used in histochemical methods (as esters) and the diazonium salt employed (Fast Blue B salt) was chosen as being the one most often used in histochemistry.

2) It was observed that the usual straight line relationship between the bimolecular velocity constant of coupling and pH (Conant and Peterson, 1930) became invalid at certain "critical points." For β-naphthol the "critical point" lay between pH 7.4 and 7.0 and for α-naphthol between pH 5.3 and 5.0.

3) Parallel histochemical studies indicated that the "critical point" and therefore the coupling rate, was likely to be important in the case of the more soluble naphthols, but of less importance with the relatively insoluble ones, in producing accurate histochemical localization of enzyme.
THE VALUE OF SOME TETRAZOLIUM SALTS AS HISTOCHEMICAL REAGENTS FOR SULPHYDRYL GROUPS
Authors: FINDLAY G. H.
Published: April 2011

A study was made of the reaction between sulphydryl compounds and tetrazolium salts, to estimate the value of the latter in the histochemistry of sulphydryl groups. The chemical reaction was found to be precarious, the relative concentrations of the two reactants making a great difference to its rate and efficiency. Hydrogen-ion concentration, oxygenation of the solutions, the presence of wetting agents and of other reducing compounds influenced the color development in a complicated manner. Mol-equivalents of various sulphydryl-containing compounds reacted differently towards tetrazolium salts. Histochemical quantitation was consequently difficult, apart from the fact that the reaction was relatively inefficient. Nevertheless, the histochemical results were considered to be comparable with those obtained with dihydroxydinaphythyldisulphide ("DDD"), although the overlap was not complete. Chemical and histochemical results were also at variance.

For histochemical purposes the method was most suited to demonstrate disulphide and sulphydryl groups together. Control sections were made in which disulphides alone were stained, and in which both sulphydryl and disulphide groups were blocked. For showing sulphydryl groups alone the reaction was least satisfactory.
THE COMPARATIVE SUSCEPTIBILITY TO COLLAGENASE AND TRYPSIN OF COLLAGEN, SOLUBLE COLLAGENS AND RENAL BASEMENT MEMBRANE
Authors: DRESNER ELLIS , SCHUBERT MAXWELL
Published: April 2011

A comparative study has been made of the susceptibility to trypsin and to collagenase of collagen and some proteins thought to be related to it. These include a crude reticulin made from embryonic calf renal midcortex and soluble collagens initially extracted from skins at alkaline or acid reactions. With minor variations all these materials are readily hydrolysed by collagenase and are rather resistant to trypsin. In all cases of hydrolysis by collagenase the solubilized portion contains hydroxyproline only in peptide form.
ON THE CHEMISTRY OF THE THIOCHOLINE METHOD OF KOELLE
Authors: MALMGREN H. , SYLVÉN B.
Published: April 2011

In the thiocholine method of Koelle as previously described (17), the enzymatic reaction step leads to the formation of crystalline deposits. These have been identified as Cu·thiocholine sulfate. The subsequent ammonium sulfide treatment aiming at the conversion of Cu·thiocholine to CuS implies that the previous crystals become dissolved and replaced by amorphous precipitates. The environmental and other operating factors in this multi-step method are discussed.
The role of exogenous electron carriers in NAD(P)-dependent dehydrogenase cytochemistry studied in vitro and with a model system of polyacrylamide films.
Authors: Van Noorden C J , Tas J
Published: April 2011

The applicability of phenazine methosulfate, 1-methoxyphenazine methosulfate, menadione, and meldola blue as exogenous electron carriers for the cytochemical staining of nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent dehydrogenases has been studied quantitatively with tetranitro BT in vitro and with a model system of polyacrylamide films incorporating either purified glucose-6-phosphate dehydrogenase or intact rat liver parenchymal cells. It was found that every assay in which a tetrazolium salt is used, whether or not an electron carrier is present, has to be carried out in darkness. Menadione did not appear to be useful, because electrons were not found to be transferred directly from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) to this compound. Phenazine methosulfate at higher concentrations and meldola blue at concentrations optimal for carrying electrons to tetrazolium salts yielded a high level of "nothing dehydrogenase" activity in cell-containing films, but no inhibition of enzymatic activity was found. Factors involved in the interference of oxygen with tetrazolium salt reduction are discussed. 1-Methoxyphenazine methosulfate did not stain cellular compounds and caused only a very low nothing dehydrogenase activity. The cytochemical demonstration of dehydrogenase activity was shown to be independent on the concentration of 1-methoxyphenazine methosulfate used (50-1000 microM). It is concluded that 1-methoxyphenazine methosulfate is the exogenous electron carrier of choice.