Use of Peroxidatic-Enzyme Staining to Enhance Resolution of Cultured Mammalian Cells Under Phase Microscopy

Staining of glutaraldehyde-fixed mammalian cells with peroxidatic enzymes (horseradish peroxidase or horse heart cytochrome c) greatly enhances resolution of their structure under phase microscopy. The topography of cell processes and regions of intercellular contact and overlapping is resolved precisely, even in dense cultures mounted in media which ordinarily do not permit clear demonstration of these areas. The technique is therefore a useful aid to the study of cultured cells with phase optics. Labeling depends on introducing free aldehydes into cells through the use of bi functional fixatives such as glutaraldehyde. Acetone or formaldehyde fixation prevents staining, and labeling intensity is greatly diminished by pretreatment with spermine, a polyamine that reacts with glutaraldehyde. Electron microscopy reveals that peroxidase tags membranes preferentially; some areas are labeled smoothly, others in a punctate manner. Ribosomes are sharply contrasted, but nuclei remain unstained. Cytochrome c labels condensed nuclear chromatin intensely, and also stains ribosomes and portions of the cyto plamic ground substance; membranes are mostly unmarked.     

Potency of microwave irradiation during fixation for electron microscopy

Summary Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl₂ and MgCl₂ led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells.Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration.We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.     

Distribution of Cytochrome c Peroxidase Activity in Wild-Type and Petite Cells of Bakers'' Yeast Grown Aerobically and Anaerobically

Studies of mitochondrial biogenesis in yeast have been hampered by a lack of suitable membrane markers in anaerobically grown cells subsequently grown in air. Cytochrome c peroxidase activity and subcellular location was studied to determine whether it would be a useful marker for an analysis of mitochondrial formation. Cytochemical tests revealed enzyme reaction product on all mitochondrial membranes in aerobically grown wild-type cells. Anaerobically grown wild-type and all petite cultures contained cytochrome c peroxidase cytochemical reaction deposits on abundant cytoplasmic membranes and on the few mitochondrial profiles which also were seen in the electron photomicrographs. Biochemical studies corroborated the cytochemistry because mitochondrial fractions were greatly enriched in cytochrome c peroxidase activity for aerobically grown wild-type cultures, but petite and anaerobically grown wild-type cultures showed higher enzyme activities in supernatant fractions than was present in the corresponding particulate fractions after differential centrifugation. Evidence from low-temperature microspectroscopy, spectrophotometric assays of mitochondrial enzyme activities, and electron microscopy showed mitochondrial formation during the time required for preparation and lysis of spheroplasts from anaerobically grown cultures. The data were interpreted as indicating that cytochrome c peroxidase was an oxygen-inducible enzyme, and that there was a developmental relationship between enzyme-reactive membranes of mitochondria and cytoplasm during the period of respiratory adaptation.     

Lateral phase separations in Escherichia coli membranes

Membranes from unsaturated fatty acid auxotrophs of Escherichia coli were studied by spin labeling and freeze-fracturing. From measurements of the partition of the spin label TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) between the aqueous phase and fluid lipids in isolated membranes, temperatures, corresponding to the onset and completion of a lateral phase separation of the membrane phospholipids were determined. By freeze-fracture electron microscopy a change in the distribution of particle in the membrane was observed around the temperature of the onset of the lateral phase separation. When cells were frozen from above that temperature a netlike distribution of particles in the plasma membrane was observed for unfixed preparations. When frozen after fixing with glutaraldehyde the particle distribution was random. In membranes of cells frozen with or without fixing from a temperature below the onset of the phase separation, the particles were aggregated and large areas void of particles were present. This behavior can be understood in terms of the freezing rate with the aid of phase diagrams.     

Potency of microwave irradiation during fixation for electron microscopy

Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.     

Tannic Acid as an Electron Microscope Tracer for Permeable Cell Membranes

To recognize damaged cells in preparations for transmission electron microscopy, high molecular weight (1700 MW) tannic acid (1-4%) has been added to glutaraldehyde fixing solutions. During fixation, the tannic acid penetrates only those cells whose plasma membranes were previously damaged. It enhances the electron density of the injured cells, which become clearly distinguishable from the undamaged ones. As a tracer tannic acid shows great advantages over either lanthanum hydroxide, ruthenium red, or horseradish peroxidase. It diffuses evenly throughout the tissue block and is not removed by preparative steps. Furthermore, it is also a good tracer at the light microscope level.     

Immunocytochemical study on the cytoplasmic side of cell membranes infected with vesicular stomatitis virus by quick-freezing and deep-etching replica method

Synthesized N protein of vesicular stomatitis virus (VSV) is associated with replicated viral genomes in the infected cells. The cytoplasmic side of cell membranes was examined by quick-freezing and deep-etching replica method, in order to clarify the localization of VSV genomes. Control or infected monolayer Vero cells were fixed in 2% paraformaldehyde, scraped and centrifuged to make pellets. A drop of the cell pellet was put between two glass coverslips, which were coated with 3-aminopropyl triethoxy silane and glutaraldehyde. The cells were consequently split open and postfixed in the mixture of glutaraldehyde and paraformaldehyde. Some inside-out cell membranes on the coverslips were immunostained with anti-N monoclonal antibody directly coupled to gold particles. Others were immunostained with anti-N monoclonal antibody and rabbit anti-mouse IgG coupled to peroxidase and fixed again in glutaraldehyde. They were incubated in diaminobenzidine and hydrogen peroxide solution for 1 min. All of them were infiltrated with 10% methanol in distilled water and quickly frozen in a mixture of isopentane and propane cooled by liquid nitrogen. Such preparations were deep-etched and shadowed by platinum and carbon. Although many cell organelles were found to be associated with the cytoplasmic side of cell membranes in the normal Vero cells, few cell organelles were attached to it in the infected cells. On the contrary, special strand structures were identified, which could be immunostained with anti-N monoclonal antibody. It is concluded that platinum replicas have sufficient resolution to identify the VSV genomes coated with N protein and that these nucleocapsids can be associated with the cytoplasmic side of cell membranes in the infected cells.     

STUDIES ON THE MODE OF ACTION OF EXCESS OF VITAMIN A : VII. Changes in the Fine Structure of Erythrocytes during Haemolysis by Vitamin A

Rabbit erythrocytes have been haemolysed by treatment with vitamin A alcohol and the sequence of changes in the fine structure of the cells during lysis has been investigated by phase contrast microscopy of intact cells and electron microscopy of thin sections. The initial effect of the vitamin, which occurs within 1 minute, is the production of cells of bizarre appearance which have a greatly increased surface area relative to untreated cells. Large indentations appear in the surfaces of the cells, and vacuoles are formed from the indentations by a process that resembles micropinocytosis. The cells then become spherical and loss of haemoglobin begins as breaks appear in the membranes of some cells; finally, ghosts are produced that are no longer spherical but still contain numerous vacuoles. These observations support the thesis that one site of action of vitamin A is at lipoprotein membranes.     

Use of cell contour analysis to evaluate the affinity between macrophages and glutaraldehyde-treated erythrocytes.

Recently, several authors evaluated the affinity between lipid bilayers or erythrocyte membranes by analyzing the deformation of cells or vesicles they brought into close contact using micromanipulators. In the present report, we extend this approach in a study of the adhesive properties of rough nucleated cells. Rat peritoneal macrophages were made to bind human red cells modified with glutaraldehyde or glutaraldehyde and polylysine. Conjugates were examined with electron microscopy, and photomicrographs were digitized for quantification of cell surface roughness in and out of adhesion areas. Also, macrophages were subjected to micropipette aspiration to find a relationship between apparent surface tension and area increase. Assuming that this increase was a direct consequence of a smoothing of the cell surface on the submicrometer scale, the actual affinity between macrophages and erythrocytes was estimated. The obtained values ranged between 8.4 X 10(-5) and 18.2 X 10(-5) J/m2. It is concluded that cell surface roughness may be an important parameter of cell adhesion and perhaps deformation. This is made amenable to experimental study by the present approach.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland     

Immunocytochemical study on the cytoplasmic side of cell membranes infected with vesicular stomatitis virus by quick-freezing and deep-etching replica method

Summary Synthesized N protein of vesicular stomatitis virus (VSV) is associated with replicated viral genomes in the infected cells. The cytoplasmic side of cell membranes was examined by quick-freezing and deep-etching replica method, in order to clarify the localization of VSV genomes. Control or infected monolayer Vero cells were fixed in 2% paraformaldehyde, scraped and centrifuged to make pellets. A drop of the cell pellet was put between two glass coverslips, which were coated with 3-aminopropyl triethoxy silane and glutaraldehyde. The cells were consequently split open and postfixed in the mixture of glutaraldehyde and paraformaldehyde. Some inside-out cell membranes on the coverslips were immunostained with anti-N monoclonal antibody directly coupled to gold particles. Others were immunostained with anti-N monoclonal antibody and rabbit anti-mouse IgG coupled to peroxidase and fixed again in glutaraldehyde. They were incubated in diaminobenzidine and hydrogen peroxide solution for 1 min. All of them were infiltrated with 10% methanol in distilled water and quickly frozen in a mixture of isopentane and propane cooled by liquid nitrogen. Such preparations were deep-etched and shadowed by platinum and carbon. Although many cell organelles were found to be associated with the cytoplasmic side of cell membranes in the normal Vero cells, few cell organelles were attached to it in the infected cells. On the contrary, special strand structures were identified, which could be immunostained with anti-N monoclonal antibody. It is concluded that platinum replicas have sufficient resolution to identify the VSV genomes coated with N protein and that these nucleocapsids can be associated with the cytoplasmic side of cell membranes in the infected cells.S. Ohno was a visiting fellow from the Fogarty International Center at the National Institutes of Health, USA, while this work was in progress     

Comparative study of conjugation between horseradish peroxidase and D-cytochrome b5. Incorporation into subcellular membranes

Horseradish peroxidase was conjugated to D-cytochrome b5 by three different two-step methods. The yield of conjugates based on the peroxidase enzymatic activity recovered after gel filtration was very low in the glutaraldehyde method, but higher in the N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and periodate methods. The molecular size of the conjugates was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monomeric conjugates were mostly formed via the glutaraldehyde and SPDP methods in the presence of appropriate molar ratios of proteins. Most of the conjugates formed via the periodate method were polymers. The conjugate preparations of the three methods could be incorporated into microsomal membranes. Conjugate polymers, however, appeared less able to be incorporated then monomers. There was a nonpreferential incorporation of free or conjugated D-cytochrome b5 contained in the conjugate preparation of the glutaraldehyde method. In conclusion, this study gives preference to the glutaraldehyde method for the preparation of conjugates that will subsequently be used as an in vivo marker of the D-cytochrome b5 incorporation into membranes.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland This revised version was published online in November 2006 with corrections to the Cover Date.     

Emperipolesis of hematopoietic cells in myelocytic leukemia. Electron microscopic and phase contrast microscopic studies

In case of blast crisis of chronic myelocytic leukemia, the blast cells contained several kinds of normal hematopoietic cells. The peroxidase reaction was strongly positive in the neutrophilic granules of the engulfed neutrophils. These engulfed cells appeared to be normal and the limiting membranes of the engulfing cells seemed to be intact. We speculated therefore that this phenomenon might be emperipolesis. In a case of chronic myelocytic leukemia and a case of acute myelocytic leukemia, some megakaryoblasts showed the same phenomenon. These megakaryoblasts did not phagocytize latex particles. The limiting membranes of the engulfing megakaryoblasts were stained with ruthenium red but those of the engulfed hematopoietic cells were not stained. By phase microscopy, the engulfed cells were actively moving inside the megakaryoblasts and it was observed that the engulfed cells were actually living within the engulfing cells. These results demonstrated that this phenomenon was emperipolesis. Observations with an electron microscope and the phase microscope are indispensable for distinguishing emperipolesis from phagocytosis.     

LacZ transgenic mice and immunoelectron microscopy: an ultrastructural method for dual localization of beta-galactosidase and horseradish peroxidase.

Transgenic animals bearing the reporter gene, LacZ, encoding the histochemical enzyme, beta-galactosidase, are increasingly becoming available. Similarly, antibody conjugates consisting of specific IgGs coupled to horseradish peroxidase (HRP) are widely used for Western blotting, ELISA, and immunohistochemistry. Here we provide a detailed fixation and histochemical protocol for the simultaneous electron microscopic visualization and discrimination of beta-galactosidase and peroxidase reaction products within mouse kidney. After incubation of transgenic LacZ tissues with IgG-HRP conjugates, samples were lightly fixed with 2% paraformaldehyde and 0.4% glutaraldehyde and processed for peroxidase histochemistry. Tissues underwent beta-galactosidase histochemistry, were refixed with glutaraldehyde, osmicated, and embedded. In Flk1/LacZ mice, we immunolocalized anti-laminin beta1 chain IgG-HRP specifically to developing glomerular basement membranes, whereas Flk1/LacZ was expressed only by glomerular endothelial cells. In Epas1/LacZ mice, we immunolocalized anti-platelet endothelial cell adhesion molecule-1 specifically to glomerular endothelial plasma membranes, whereas Epas1/LacZ was expressed by both glomerular endothelial and mesangial cells. This dual ultrastructural localization technique should be broadly applicable for immunoelectron microscopic studies in LacZ transgenic animals, particularly those where LacZ expression and antibody-HRP binding are both relatively abundant.     

THE FINE STRUCTURAL LOCALIZATION OF ENDOGENOUS AND EXOGENOUS PEROXIDASE ACTIVITY IN KUPFFER CELLS OF RAT LIVER

Endogenous peroxidase activity has been demonstrated in sections of rat liver fixed briefly by glutaraldehyde perfusion and incubated in Graham and Karnovsky's medium for cytochemical demonstration of peroxidase activity (29). In 25–40% of sinusoidal cells, an electron-opaque reaction product is localized in segments of the endoplasmic reticulum, including the perinuclear cisternae, a few Golgi vesicles and saccules and in some large membrane-bounded granules. This staining is abolished after prolonged fixation or boiling of tissue sections in glutaraldehyde, and in the absence of H₂O₂ or DAB from the incubation medium. Furthermore, the reaction is inhibited completely by sodium azide and high concentrations of H₂O₂, and partially by KCN and aminotriazole. Among the different cells in hepatic sinusoids, the nonphagocytic "fat-storing" cells (39) are always peroxidase negative, whereas the lining cells in process of erythrophagocytosis are consistently peroxidase positive. The possible biological significance of endogenous peroxidase in Kupffer cells is discussed. In addition, the uptake of exogenous horseradish peroxidase by Kupffer cells has been investigated. The exogenous tracer protein, which in contrast to endogenous peroxidase of Kupffer cells is not inhibited by prolonged aldehyde fixation, is taken up by micropinocytosis and remains confined to the lysosomal system of Kupffer cells. The significance of these observations in respect to some recent studies suggesting localization of exogenous peroxidases in the endoplasmic reticulum of Kupffer cells and peritoneal macrophages (22, 23) is briefly discussed.     

Cell membrane coating with glutaraldehyde: application to a versatile solid-phase assay for thyroid membrane proteins and molecules interacting with thyroid membranes

In defined conditions, glutaraldehyde was shown to tightly bind cell membranes to flexible microtiter plates without significant alteration of the antigenic and functional properties of membrane proteins. In the presence of 0.06% glutaraldehyde, human thyroid membranes were bound to plastic firmly enough to resist numerous washing and flicking steps; the coated membranes remained almost unaltered with regard to monoclonal antibody and thyrotropin binding as well as adenylate cyclase and peroxidase activities. Based on the use of thyroid membrane-coated microtiter plates, a versatile solid-phase assay was developed which allowed screening of anti-membrane monoclonal antibodies, detection of thyrotropin-displacing activity in hormone and antibody preparations, and monitoring of fractionation experiments of solubilized membrane antigens and thyrotropin receptor. It was concluded that the use of glutaraldehyde for coating cell membranes to flexible microtiter plates enabled the establishment of simple, rapid, and reliable assays for detection and quantitation of membrane proteins and molecules interacting with membranes.     

Membrane formation by interfacial cross-linking of chitosan for microencapsulation of Lactococcus lactis

Lactic acid bacteria were microencapsulated within cross-linked chitosan membranes formed by emulsification/interfacial polymerization. The technique was modified and optimized to provide biocompatible conditions during encapsulation involving the use of mineral oils as the continuous phase and chitosan as the membrane material. Chitosan cross-linked with hexamethylene diisocyanate or glutaraldehyde resulted in strong membranes, with a narrow size distribution about a mean diameter of 150 mum. Cell viability and activity was demonstrated by the acidification of milk. Loss of acidification activity during microencapsulation was recovered in subsequent fermentations to levels similar to that of free cell fermentations. (c) 1993 John Wiley & Sons, Inc.     

Structure of the nucleoid in cells of Streptococcus faecalis

The structure of the nucleoid of Streptococcus faecalis (ATCC 9790) was examined and compared in the unfixed and fixed states by immersive refractometry and electron microscopy. It appears from these studies that the nucleoid structure is much more centralized in unfixed chloramphenicol-treated (stationary-phase) cells than it is in cells in the exponential phase of growth. The more dispersed configuration of the exponential-phase nucleoid could be preserved by fixation in glutaraldehyde, but not in Formalin or in osmium tetroxide. One important factor in explaining these differences in preservation is that glutaraldehyde (but not Formalin or osmium tetroxide) can rapidly cross-link the amino groups of macromolecules in cells. It was also observed that osmium tetroxide resulted in a preferential breakdown of nascent ribonucleic acid. These results are interpreted as indicating that glutaraldehyde is able to stabilize the exponential-phase nucleoid before it assumes the more central appearance seen in osmium tetroxide- and Formalin-fixed cells. These results are discussed in terms of the proposed organization of the exponential-phase nucleoid in unfixed cells.