Postembedding immunogold labeling for electron microscopy using ?LR White? resin

We have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1-μm sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning.

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Comparison of LR White and Unicryl as embedding media for light and electron immunomicroscopy of chromaffin cells.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.3.8648090 ◽

1996 ◽

Vol 44 (3) ◽

pp. 289-295 ◽

Cited By ~ 12

Author(s):

G Goping ◽

G A Kuijpers ◽

R Vinet ◽

H B Pollard

Keyword(s):

Electron Microscopy ◽

Tyrosine Hydroxylase ◽

Membrane Fusion ◽

Chromaffin Cells ◽

Chromaffin Cell ◽

Osmium Tetroxide ◽

Immunogold Labeling ◽

Dopamine Beta Hydroxylase ◽

Lr White ◽

Embedding Medium

LR White and Unicryl are members of the same family of acrylic embedding resins and are very suitable for "on grid" postembedding immunogold labeling. We studied the ultrastructure of LR White- and Unicryl-embedded cultured chromaffin cells and the immunolocalization of three chromaffin cell proteins, the enzymes dopamine-beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH), and the membrane fusion and Ca2+ channel protein synexin (annexin VII). We report here that Unicryl is preferable to LR White as an embedding medium for electron microscopy when osmium tetroxide fixation is omitted. The basis for this distinction is better ultrastructural preservation and improved immunodetection efficiency.

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Immunogold labeling of Pneumocystis carinii for Electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100085654 ◽

1991 ◽

Vol 49 ◽

pp. 270-271

Author(s):

Michael P. Goheen ◽

Marilyn S. Bartlett ◽

James W. Smith

Keyword(s):

Electron Microscopy ◽

Pneumocystis Carinii ◽

Severe Pneumonia ◽

Culture Fluid ◽

Immunogold Labeling ◽

Antigenic Sites ◽

Transmission Electron ◽

Response To Chemotherapy ◽

Acquired Immunodeficiency ◽

Immunodeficiency Syndrome

Studies of the biology of Pneumocystis carinii (PC) are of increasing importance because this extracellular pathogen is a frequent source of severe pneumonia in patients with acquired immunodeficiency syndrome (AIDS) and is a leading cause of mortality in these patients. Immunoelectron microscopic localization of antigenic sites on the surface of PC would improve the understanding of these sites and their role in pathenogenisis of the disease and response to chemotherapy. The purpose of this study was to develop a methodology for visualizing immunoreactive sites on PC with transmission electron microscopy (TEM) using immunogold labeled probes.Trophozoites of PC were added to spinner flask cultures and allowed to grow for 7 days, then aliquots of tissue culture fluid were centrifuged at 12,000 RPM for 30 sec. Pellets of organisims were fixed in either 1% glutaraldehyde, 0.1% glutaraldehyde-4% paraformaldehyde, or 4% paraformaldehyde for 4h. All fixatives were buffered with 0.1M Na cacodylate and the pH adjusted to 7.1. After fixation the pellets were rinsed in 0.1M Na cacodylate (3X), dehydrated with ethanol, and immersed in a 1:1 mixture of 95% ethanol and LR White resin.

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Factors influencing adhesive bonding at the bone/implant interface

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100130201 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1114-1115

Author(s):

Julie A. Martini ◽

Robert H. Doremus

Keyword(s):

Electron Microscopy ◽

Adhesive Bonding ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Bone Implant ◽

Factors Influencing ◽

Lr White ◽

Transmission Electron ◽

New Zealand White Rabbits ◽

Scanning Electron

Tracy and Doremus have demonstrated chemical bonding between bone and hydroxylapatite with transmission electron microscopy. Now researchers ponder how to improve upon this bond in turn improving the life expectancy and biocompatibility of implantable orthopedic devices.This report focuses on a study of the- chemical influences on the interfacial integrity and strength. Pure hydroxylapatite (HAP), magnesium doped HAP, strontium doped HAP, bioglass and medical grade titanium cylinders were implanted into the tibial cortices of New Zealand white rabbits. After 12 weeks, the implants were retrieved for a scanning electron microscopy study coupled with energy dispersive spectroscopy.Following sacrifice and careful retrieval, the samples were dehydrated through a graduated series starting with 50% ethanol and continuing through 60, 70, 80, 90, 95, and 100% ethanol over a period of two days. The samples were embedded in LR White. Again a graduated series was used with solutions of 50, 75 and 100% LR White diluted in ethanol.

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Immunogold labeling in scanning electron microscopy

Histochemistry and Cell Biology ◽

10.1007/bf02473200 ◽

1996 ◽

Vol 106 (1) ◽

pp. 31-39 ◽

Cited By ~ 71

Author(s):

R. Hermann ◽

P. Walther ◽

M. Müller

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Immunogold Labeling ◽

Scanning Electron

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Immunological discrimination between a sap-staining fungus and a biological control fungus

Canadian Journal of Botany ◽

10.1139/b90-203 ◽

1990 ◽

Vol 68 (7) ◽

pp. 1578-1588 ◽

Cited By ~ 4

Author(s):

Brian T. Luck ◽

Colette Breuil ◽

David L. Brown

Keyword(s):

Electron Microscopy ◽

Biological Control ◽

Immunosorbent Assay ◽

Liquid Culture ◽

Biological Control Agent ◽

Dry Weight ◽

Cross Reactivity ◽

Immunogold Labeling ◽

Enzyme Linked Immunosorbent Assay ◽

Polyclonal Serum

An enzyme-linked immunosorbent assay (ELISA) was used to detect a sap-staining fungus, Ophiostoma piceae, and a biological-control agent, Gliocladium roseum, grown in liquid culture and in wood. A polyclonal serum prepared against whole cell fragments from broken mycelia of O. piceae detected O. piceae in liquid culture at 0.25 μg dry weight/mL; however, there was moderate cross-reactivity with G. roseum. Antiserum adsorbed on G. roseum had almost no reactivity with G. roseum but still reacted strongly with O. piceae. The specificity of these sera was verified, and the antigenic sites were localized, by immunogold labeling and electron microscopy. These studies confirmed that the adsorbed serum could differentiate between G. roseum and O. piceae and showed that the cell wall was the most reactive cellular component. These results are discussed in relation to the development of immunological probes for the detection of sap-staining and biological control fungi. Key words: polyclonal serum, enzyme-linked immunosorbent assay, immunogold labeling, sap-staining and biological control fungi, electron microscopy.

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A Novel Flat-embedding Method to Prepare Ultrathin Cryosections from Cultured Cells in Their In Situ Orientation

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540205000809 ◽

2002 ◽

Vol 50 (8) ◽

pp. 1067-1080 ◽

Cited By ~ 42

Author(s):

Viola Oorschot ◽

Heidi de Wit ◽

Wim G. Annaert ◽

Judith Klumperman

Keyword(s):

Electron Microscopy ◽

Quantitative Method ◽

Cultured Cells ◽

Petri Dish ◽

Ultrastructural Level ◽

Immunogold Labeling ◽

Polarized Cells ◽

Ultrathin Cryosections ◽

Flat Embedding

Immunogold labeling of ultrathin cryosections provides a sensitive and quantitative method to localize proteins at the ultrastructural level. An obligatory step in the routine preparation of cryosections from cultured cells is the detachment of cells from their substrate and subsequent pelleting. This procedure precludes visualization of cells in their in situ orientation and hampers the study of polarized cells. Here we describe a method to sample cultured cells from a petri dish or coverslip by embedding them in a 12% gelatin slab. Subsequently, sections can be prepared in parallel or perpendicular to the plane of growth. Our method extends the cryosectioning technique to applications in studying polarized cells and correlative light–electron microscopy.

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