Freeze-substitution as a preparative technique for immunoelectronmicroscopy: Evaluation by atomic force microscopy

Moreira, Jorge E.; Reese, Thomas S.; Kachar, Bechara

doi:10.1002/(sici)1097-0029(19960215)33:3<251::aid-jemt2>3.0.co;2-t

Cited by 16 publications

(11 citation statements)

References 23 publications

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“…Samples of axoplasm for structural comparison were prepared by substituting them at Ϫ80C with osmium tetroxide in acetone containing 4% osmium tetroxide for 36 hr (Moreira et al 1996). Samples were then passively warmed to Ϫ20C, transferred to crushed ice for 1 hr, block-stained with 1% uranyl acetate in acetone, rinsed briefly in methanol, substituted with propylene oxide, and embedded in Araldite resin (CY212).…”

Section: Methodsmentioning

confidence: 99%

“…Axoplasm prepared by conventional freeze-substitution (Moreira et al 1996) could be examined up to 10-15 m from the freezing surface, where it disappeared into ice crystal patterns. Axoplasm substituted with osmium was similar to that in axons fixed by immersion in glutaraldehyde and postfixed with osmium (see Miller and Lasek 1985) in showing a thick mat of indistinct cytoskeletal elements, among which only microtubules and membranes were clearly differentiated ( Figure 1A).…”

Section: Axoplasmmentioning

confidence: 99%

See 1 more Smart Citation

Immunoelectronmicroscopy of Soluble and Membrane Proteins with a Sensitive Postembedding Method

Moreira

Dodane

Reese

1998

J Histochem Cytochem.

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SUMMARYThe application of immunoelectronmicroscopy to soluble proteins is limited because soluble proteins can redistribute during fixation. Fixation may also adversely affect the recognition of proteins associated with membranes. We show here how displacements of soluble proteins can be prevented and antigen sensitivity improved by freeze-substitution immunocytochemistry. The usefulness of this method for soluble cytoplasmic proteins is demonstrated for the twitchin protein in Aplysia muscle and the kinesin motor proteins in squid giant axons, in which the sizes of various cytoplasmic pools of kinesins are estimated. The utility for membrane proteins present in small numbers of copies is demonstrated by labeling a glutamate receptor subunit in mouse cerebellar cortex and the ZO-1 protein in tight junctions between MDCK cells. Thus, freeze-substitution immunocytochemistry can show the native distribution of both soluble and membrane proteins labeled with polyclonal antibodies and, at the same time, can reveal structural features comparable to those in chemically fixed or osmium freeze-substituted samples.

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Section: Methodsmentioning

confidence: 99%

Section: Axoplasmmentioning

confidence: 99%

Immunoelectronmicroscopy of Soluble and Membrane Proteins with a Sensitive Postembedding Method

Moreira

Dodane

Reese

1998

J Histochem Cytochem.

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“…MePD samples were rapidly submerged in Freon cooled by liquid nitrogen, then immersed in 1.5% uranyl acetate in methanol for 24 hours at 290 C in an automatic freezesubstitution system unit (Leica EM-AFS, Leica Microsystems). The temperature was increased in steps of 4 C/h from 290 C to 245 C and samples were rinsed in methanol and infiltrated with Lowicryl HM20 resin (EMS) using increasing concentrations of Lowicryl (60%, 75% and 100%) in methanol for 48 hours at 245 C. Blocks were polymerized with UV light (360 nm) at an initial temperature of 245 C, increasing 1 C/h until RT (Moreira et al, 1996(Moreira et al, , 1998Zhao et al, 1998). Blocks were trimmed for semithin (500 nm) and ultrathin (70 nm) sections, and placed on nickel grids (200 hexagonal mesh SynapTek EMS) or single slot grids (SynapTek, 1 3 2 mm, EMS) coated with Formvar (EMS) for serial sections.…”

Section: Immunogold Electron Microscopymentioning

confidence: 99%

Inhibitory and multisynaptic spines, and hemispherical synaptic specialization in the posterodorsal medial amygdala of male and female rats

Brusco

Merlo

Ikeda

et al. 2014

J of Comparative Neurology

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The density of dendritic spines is sexually dimorphic and variable throughout the female estrous cycle in the rat posterodorsal medial amygdala (MePD), a relevant area for the modulation of reproductive behavior in rats. The local synaptic activity differs between hemispheres in prepubertal animals. Here, we used serial section transmission electron microscopy to produce three-dimensional reconstructions of dendritic shafts and spines to characterize synaptic contacts on MePD neurons of both hemispheres in adult males and in females along the estrous cycle. Pleomorphic spines and non-synaptic filopodia occur in the MePD. On average, 8.6% of dendritic spines received inputs from symmetric GABA-immunoreactive terminals, whereas 3.6% received two synaptic contacts on the spine head, neck or base. Presynaptic terminals in females right MePD had a higher density of synaptic vesicles and docked vesicles than the left MePD, suggesting a higher rate of synaptic vesicle release in the right MePD of female rats. In contrast, males did not show laterality in any of those parameters. The proportion of putative inhibitory synapses on dendritic shafts in the right MePD of females in proestrus was higher than in the left MePD, and higher than in the right MePD in males, or in females in diestrus or estrus. This work shows synaptic laterality depending on sex and the estrous cycle phases in mature MePD neurons. Most likely, sexual hormones effects are lateralized in this brain region, leading to higher synaptic activity in the right than in the left hemisphere of females, mediating timely neuroendocrine and social/reproductive behavior.

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“…Procedures were performed as described by Moreira et al (1996). In brief, rat aorta treated or not treated with CD (10 mM) for 60 min was dissected out and fixed by immersion in a solution containing 2% glutaraldehyde and 2% paraformaldehyde in 0.1 mM sodium cacodylate buffer for 24 h. Preparations were kept in sodium cacodylate (0.1 mM) in a freezer until the next step.…”

Section: Electron Microscopymentioning

confidence: 99%

Caveolae Dysfunction Contributes to Impaired Relaxation Induced by Nitric Oxide Donor in Aorta from Renal Hypertensive Rats

Rodrigues

Restini

Lunardi

et al. 2007

The Journal of Pharmacology and Experimental Therapeutics

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Relaxation induced by nitric oxide (NO) donors is impaired in renal hypertensive two kidney-one clip (2K-1C) rat aortas. It has been proposed that caveolae are important in signal transduction and Ca 2ϩ homeostasis. Therefore, in the present study we investigate the integrity of caveolae in vascular smooth muscle cells (VSMCs), as well as their influence on the effects produced by NO released from both the new NO donor [Ru(NH.NHq) (terpy)NO ϩ ] 3ϩ (TERPY) and sodium nitroprusside (SNP) on 2K-1C rat aorta. The potency of both TERPY and SNP was lower in the 2K-1C aorta that in the normotensive aorta [two kidney (2K)], whereas the maximal relaxant effect (ME) was similar in both 2K-1C and 2K aortas. In the 2K aorta, methyl-␤-cyclodextrin (CD) reduced both the potency of TERPY and SNP, and their ME compared with the control, but it had no effect on the potency and ME of these NO donors in 2K-1C aortas. The decrease in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] c ) induced by TERPY was larger in 2K than in 2K-1C cells, and this effect was inhibited by CD in 2K cells only. Aortic VSMCs from 2K rats presented a larger number of caveolae than those from 2K-1C rats. Treatment with CD reduced the number of caveolae in both 2K and 2K-1C aortic VSMCs. Our results support the idea that caveolae play a critical role in the relaxant effect and in the decrease in [Ca 2ϩ

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Freeze-substitution as a preparative technique for immunoelectronmicroscopy: Evaluation by atomic force microscopy

Cited by 16 publications

References 23 publications

Immunoelectronmicroscopy of Soluble and Membrane Proteins with a Sensitive Postembedding Method

Immunoelectronmicroscopy of Soluble and Membrane Proteins with a Sensitive Postembedding Method

Inhibitory and multisynaptic spines, and hemispherical synaptic specialization in the posterodorsal medial amygdala of male and female rats

Caveolae Dysfunction Contributes to Impaired Relaxation Induced by Nitric Oxide Donor in Aorta from Renal Hypertensive Rats

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