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

2002 ◽  
Vol 50 (8) ◽  
pp. 1067-1080 ◽  
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.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

scholarly journals A Quantitative Method for In-Situ Pump-Beam Metrology in Ultrafast Electron Microscopy

2021 ◽  
Vol 27 (S1) ◽  
pp. 3416-3418
Author(s):  
Jialiang Chen ◽  
Chris Leighton ◽  
David Flannigan
Keyword(s):  
Electron Microscopy ◽  
Pump Beam ◽  
Quantitative Method

scholarly journals Simplified in situ preparation of cultured cell monolayers for electron microscopy.

1980 ◽  
Vol 28 (2) ◽  
pp. 178-180 ◽  
Author(s):  
K Kawamoto ◽  
A Hirano ◽  
F Herz
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Growth Surface ◽  
Cultured Cell ◽  
Cell Monolayers ◽  
In Situ Preparation

The use of xylene for the easy separation of cultured cells embedded in situ from their plastic growth surface is described. This step simplifies the preparation of cell monolayers for electron microscopy.

Fluoronanogold as a Probe for High Resolution Correlation Between ImmunoFluorescence and Electron Microscopy

1999 ◽  
Vol 5 (S2) ◽  
pp. 476-477
Author(s):  
T. Takizawa ◽  
J. M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Immunofluorescence Microscopy ◽  
Human Neutrophils ◽  
Secondary Antibody ◽  
Marker Protein ◽  
Fab Fragment ◽  
Ultrathin Cryosections

[Introduction] Immunocytochemical labeling of cryosections, especially immunofluorescence microscopy using semi-thin (0.5-μm) cryosections, has been a powerful technique for detection of cellular antigens in situ and has been widely employed in cell and molecular biology studies. In many cases, immunofluorescence provides sufficient resolution and sensitivity to answer the question being addressed. However, in certain cases the increased resolution of the electron microscope using ultrathin (90-nm) cryosections may be required to define more precisely the localization of specific molecules. Recently, a unique fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), has been developed for use as a secondary antibody in immunocytochemical applications. It consists of a Fab' fragment of an antibody to which a 1.4-nm gold particle and fluorochromes are conjugated. FNG permits correlative microscopic observation of a sample stained in a single labeling procedure by multiple optical imaging. Recently, we have shown FNG immunocytochemistry on ultrathin cryosections to be valuable for high-resolution correlation of immunofluorescence and immunoelectron microscopy. In the present study, we have examined the utility of FNG as a secondary antibody for immunolabeling of myeloperoxidase (a marker protein for the azurophillic granules) in ultrathin cryosectioned human neutrophils.[Materials and Methods] Purified human neutrophils were fixed with paraformaldehyde, embedded in gelatin, infiltrated with sucrose, cut as ultrathin cryosections, and then collected on formvar film-coated nickel EM grids as described previously. Grids containing ultrathin cryosections were incubated with antimyeloperoxidase and then incubated with FNG.

scholarly journals Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy.

1987 ◽  
Vol 35 (8) ◽  
pp. 843-853 ◽  
Author(s):  
G B Birrell ◽  
K K Hedberg ◽  
O H Griffith
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Light Microscopy ◽  
Tannic Acid ◽  
Cultured Cells ◽  
Immunogold Labeling ◽  
Fish Gelatin ◽  
Nonspecific Binding ◽  
Gold Particles ◽  
Transmission Electron

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.

scholarly journals Immunogold Labeling of Cultured Cells and Virus Particles for Electron Microscopy and Cryo-Electron Microscopy Applications.

2014 ◽  
Vol 20 (S3) ◽  
pp. 1220-1221
Author(s):  
Hong Yi ◽  
Joshua D. Strauss ◽  
Jason E. Hammonds ◽  
Ravi Dyavar Shetty ◽  
Rama R. Amara ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Immunogold Labeling ◽  
Virus Particles ◽  
Cryo Electron Microscopy

scholarly journals Immunogold electron microscopy of phytochrome in Avena: identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability.

1986 ◽  
Vol 103 (6) ◽  
pp. 2541-2550 ◽  
Author(s):  
D W McCurdy ◽  
L H Pratt
Keyword(s):  
Electron Microscopy ◽  
Red Light ◽  
Freeze Substitution ◽  
Immunogold Labeling ◽  
Immunogold Electron Microscopy ◽  
Subcellular Component ◽  
Discrete Structures

Using monoclonal antibodies to the plant photoreceptor, phytochrome, we have investigated by immunogold electron microscopy the rapid, red light-induced, intracellular redistribution (termed "sequestering") of phytochrome in dark-grown Avena coleoptiles. Pre-embedding immunolabeling of 5-micron-thick cryosections reveals that sequestered phytochrome is associated with numerous, discrete structures of similar morphology. Specific labeling of these structures was also achieved by post-embedding ("on-grid") immunostaining of LR-White-embedded tissue, regardless of whether the tissue had been fixed chemically or by freeze substitution. The phytochrome-associated structures are globular to oval in shape, 200-400 nm in size, and are composed of amorphous, granular material. No morphologically identifiable membranes are present either surrounding or within these structures, which are often present as apparent aggregates that approach several micrometers in size. An immunogold labeling procedure has also been developed to identify the particulate, subcellular component with which phytochrome is associated in vitro as a consequence of irradiation of Avena coleoptiles before their homogenization. Structures with appearance similar to those identified in situ are the only components of the pelletable material that are specifically labeled with gold. We conclude that the association of phytochrome with these structures in Avena represents the underlying molecular event that ultimately is expressed both as red light-induced sequestering in vivo and enhanced pelletability of phytochrome detected in vitro.

Sign in / Sign up

Export Citation Format

Share Document