Methods of Preparation for Electron Microscopy

Robinson, David G.; Ehlers, Ulrich; Herken, Rainer; Herrmann, Bernd; Mayer, Frank; Schürmann, Friedrich‐Wilhelm

doi:10.1007/978-3-642-48848-1

Cited by 95 publications

(59 citation statements)

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Section: Methodsmentioning

confidence: 99%

“…Osmium fixation was done with 1% (wt/vol) OsO 4 in Veronal-sodium acetate buffer, pH 7.0, overnight at 4ЊC. The fruiting bodies were further processed according to the general protocol for epoxy resins (15,18).Electron microscopy of ultrathin sections. Ultrathin sections of 120 nm thickness for electron microscopy were made with a Reichert Ultracut S ultramicrotome (Leica, Bensheim, Germany) and mounted on carbon-coated Formvar grids.…”

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confidence: 99%

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Localization of the stress protein SP21 in indole-induced spores, fruiting bodies, and heat-shocked cells of Stigmatella aurantiaca

1995

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The localization and distribution of the stress protein SP21 in indole-induced vegetative cells, fruiting bodies, and heat shocked cells of Stigmatella aurantiaca were determined by immunoelectron microscopy. SP21 was found at the cell periphery in heat-shocked cells and either at the cell periphery or within the cytoplasm in indole-induced cells, often concentrated in clusters. In fruiting-body-derived spores, SP21 was located mainly at the cell wall, preferentially at the outer periphery. Furthermore, SP21 antigen was associated with cellular remnants within the stalk and within the peripheral horizon next to the fruiting body.Stigmatella aurantiaca is a gram-negative, rod-shaped myxobacterium that grows on decaying organic matter in soil. The myxobacteria possess a biphasic cell cycle, in the course of which they form a fruiting body.During vegetative growth, the cells glide in swarms upon insoluble organic substrates which they partially degrade by means of secreted lytic enzymes. Cells that live in a dense swarm accumulate a high local concentration of enzymes which provide the cells with a higher concentration of soluble metabolites and efficiently prevent these metabolites from escaping (2). When nutrients are depleted, the cells migrate into aggregation centers, from which the fruiting bodies arise. The fruiting body of S. aurantiaca consists of a branched stalk supporting the sporangioles, which in turn contain several thousands of myxospores. During fruiting-body formation, large amounts of slimes are produced and there is a concomitant massive cell lysis (22). Within the sporangiole, cells convert into myxospores as resting cells with increased resistance to environmental stresses, such as heat, UV light, and desiccation.Features observed during eukaryotic multicellular morphogenesis, such as the processing of positional information and cell communication by close contact or diffusible molecules, are predicted to play an important role in fruiting of the myxobacteria (17).Spore formation of S. aurantiaca is not strictly coupled to fruiting-body formation and can be directly induced in liquid culture by a number of chemicals, of which the most potent are indole and some of its derivatives (3).During development, a protein with an M r of 21,000 (SP21) which was found in fruiting bodies and in indole-induced spores but was not detected in vegetative cells growing under standard conditions is synthesized (5). Stress conditions such as heat shock or limitation of oxygen also induce the synthesis of SP21. Sequence homology of the polypeptide sequence deduced from the corresponding gene (hspA) identifies SP21 as a member of the family of low-molecular-weight heat shock proteins (LMW-HSP). The predicted polypeptide is more similar to some plant LMW-HSP (53% identity) than it is to any of the known bacterial LMW-HSP (4).As SP21 was sedimented in the membrane fraction of cell lysates, we wanted to know if the protein is associated with the membranes or if it is part of a high-molecular-weight complex. Immunoele...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Localization of the stress protein SP21 in indole-induced spores, fruiting bodies, and heat-shocked cells of Stigmatella aurantiaca

1995

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“…Negative staining was performed on carbon-coated mica, as described previously (Robinson et al, 1987). Immunogold negative staining was performed as described by Drucker et al (1995).…”

Section: Negative Staining and Immunogold Negative Stainingmentioning

confidence: 99%

In Situ Localization and in Vitro Induction of Plant COPI-Coated Vesicles

et al. 2000

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Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis -face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis -Golgi stack, gradually diminishing toward the trans -Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/ Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley ␣ -amylase-HDEL yielded a COPI-coated vesicle fraction that contained ␣ -amylase as well as calreticulin. INTRODUCTIONProtein trafficking in the secretory and endocytotic pathways is facilitated by coated vesicles (Rothman and Wieland, 1996;Robinson et al., 1998). Research on mammalian and yeast cells has established that two different types of non-clathrin-coated vesicles are responsible for transport events occurring between the endoplasmic reticulum (ER) and the Golgi apparatus and for intra-Golgi transport. Whereas investigators generally agree that COPIIcoated vesicles bud from the ER and transport proteins in the anterograde direction (Barlowe et al., 1994;Bannykh and Balch, 1998), the function of COPI-coated vesicles remains controversial. Strong evidence suggests that COPIcoated vesicles are responsible for the recycling of escaped ER-resident proteins from post-ER compartments (Letourneur et al., 1994), but data have also been presented supporting a role in anterograde transport of proteins, especially within the Golgi stack (Orci et al., 1997;Lippincott-Schwartz et al., 1998).The formation of both types of COP-coated vesicles involves the recruitment of a coat protein complex (coatomer for COPI-coated vesicles, Sec23/24 and Sec13/31 dimers for COPII-coated vesicles) by a membrane-associated GTP binding protein (Arf1p and Sar1p for COPI and COPII, respectively; Schekman and Orci, 1996). Both Arf1p and Sar1p exist in the cytosol as GDP-bound forms and become converted to the GTP form through the action of a membrane-associated exchange factor, guanine nucleotide exchange factor (Helms and Rothman, 1992). For Sar1p, this is Sec12p, a transmembrane protein of the ER ; for Arf1p, the most likely candidates are Gea1/2p and ARNO (ARF nucleotide binding site opener; ...

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“…Some specimens were cut with freeze fracture after immersion in liquid nitrogen. The testes of all animals were examined using scanning electron microscope Philips XL30 (Robinson et al, 1987).…”

Section: Methodsmentioning

confidence: 99%

An Electron Microscopic Study of the Antifertility Potential of Rosemary (Rosmarinus officinalis L.) in Male Albino Rats

2012

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EL-DIN, R. A. S.; EL-SHAHAT, A. E. & ELMANSY, R.A. An electron microscopic study of the antifertility potential of Rosemary (Rosmarinus officinalis L.) in male albino rats. Int. J. Morphol., 30(2):666-672, 2012. SUMMARY:The present work was aimed at studying the antifertility potential of the commonly used herb, rosemary in the male albino rats using electron microscopy as the method of investigation. Ethanolic extract of the rosmary prepared and administered orally in two different doses for a period of three months to the animals. At the end of the experiment animals were sacrificed and testes removed. Sections for the electrone microscopy prepared and changes were observed. The present results showed evident microscopic changes in the testis of the animals received higher dose of the drug. Most of the seminiferous tubules were compressed, having irregular basement membrane and devoid of any spermatogenic cells. The present work revealed a clear morphological evidence of the dose dependent antifertility potential of the rosemary in the male albino rats.

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Methods of Preparation for Electron Microscopy

Cited by 95 publications

References 2 publications

Localization of the stress protein SP21 in indole-induced spores, fruiting bodies, and heat-shocked cells of Stigmatella aurantiaca

Localization of the stress protein SP21 in indole-induced spores, fruiting bodies, and heat-shocked cells of Stigmatella aurantiaca

In Situ Localization and in Vitro Induction of Plant COPI-Coated Vesicles

An Electron Microscopic Study of the Antifertility Potential of Rosemary (Rosmarinus officinalis L.) in Male Albino Rats

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