High-pressure freezing combined with in vivo-DAB-cytochemistry: A novel approach for studies of endocytic compartments

Ellinger, Adolf; Vetterlein, M.; Weiss, Christoph; Meisslitzer-Ruppitsch, Claudia; Neumüller, Josef; Pavelka, Margit

doi:10.1016/j.jsb.2009.10.011

Cited by 11 publications

(10 citation statements)

References 59 publications

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“…In addition to DAB cytochemistry after HPF/FS and sample rehydration, we optionally cross linked Tf‐HRP with DAB in vivo by adapting methods published by others (i.e. DAB cross linking in vivo for 4–5 min at room temperature , followed by HFP and FS [with glutaraldehyde‐based, low‐OsO 4 FS media) and epoxy resin embedding ].…”

Section: Methodsmentioning

confidence: 99%

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System

Vogel

Ebner

Araújo

et al. 2015

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Section: Methodsmentioning

confidence: 99%

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System

Vogel

Ebner

Araújo

et al. 2015

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“…We would like to emphasize that EM analyses of PCs significantly supplement the routinely used quality assessment methods monitoring different manufacturing procedures. Methodical refinements, improving the preparation protocols of EM specimens, such as a wide range of cryomethods, stabilizing the samples close to the living state, together with electron tomography, 3D-reconstruction and modeling visualize structural details and dynamic processes at high resolution [99,147,148]. Thus new in vitro and in vivo tests of PLT function in transfused PCs can be efficiently controlled at the EM level.…”

Section: The Transmission Electron Microscope -Theory and Applicationsmentioning

confidence: 99%

Transmission Electron Microscopy of Platelets FROM Apheresis and Buffy-Coat-Derived Platelet Concentrates

Neumüller¹,

Ellinger²

2015

The Transmission Electron Microscope - Theory and Applications

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Platelet concentrates are produced in order to treat bleeding disorders. They can be provided by apheresis machines or by pooling of buffy coats from four blood donations. During their manufacturing and storage, morphological alterations of platelets occur which can be demonstrated by transmission electron microscopy. Alterations range from slight and reversible changes, such as formation of small cell protrusions and swelling of the surface-connected open canalicular system, to severe structural changes, where platelets undergo transitions from discoid to ameboid shapes as a consequence of platelet activation. These alterations end in delivery of the contents of platelet granules as well as platelet involution caused by apoptosis and necrosis processes denoted as the platelet release reaction. Hereby, the involvement of the network of the open canalicular system, helping to deliver the contents of platelet granules into the surrounding milieu via pores, is distinctly shown by electron tomography. As a consequence of platelet activation, a delivery of differently sized microparticles takes place which is thought to play an important role in the adverse reactions in some recipients of platelet concentrates. In this article, the formation and delivery of platelet microparticles is illustrated by electron tomography. Above all, the ultrastructural features of platelets and megakaryocytes are discussed in the context of the molecular characteristics of the plasma membrane and organelles including the different granules and the expression of receptors engaged in signaling during platelet activation. Starting from the knowledge of the ultrastructure of resting and activated platelets, a score classification is presented, allowing the evaluation of different activation stages in a reproducible manner. Examples of evaluations of platelet concentrates using electron microscopy are briefly reviewed. In the last part,

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“…In addition to its much better photo-physical properties, fluorescence imaging of BChol can be combined with electron microscopy studies making use of the diaminobenzidine reaction [105]. Upon illumination of BChol in glutaraldehyde or formaldehyde fixed cells, reactive oxygen species cause photo-oxidation of diaminobenzidine, thereby creating an electron-dense precipitate in sub-cellular regions where BChol is located [105, 106]. By this method, BChol was found in tubular endosomes, multivesicular bodies and the trans-Golgi network in human HepG2 hepatoma cells.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Cholesterol Trafficking with Fluorescent Probes

Maxfield

Wüstner

2012

Methods in Cell Biology

213

195

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Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly.

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High-pressure freezing combined with in vivo-DAB-cytochemistry: A novel approach for studies of endocytic compartments

Cited by 11 publications

References 59 publications

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System

Transmission Electron Microscopy of Platelets FROM Apheresis and Buffy-Coat-Derived Platelet Concentrates

Analysis of Cholesterol Trafficking with Fluorescent Probes

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