Lectin-Induced RBC Agglutination: Chemical vs. Cryofixation

1980 ◽  
Vol 38 ◽  
pp. 664-665
Author(s):  
Dean A. Handley ◽  
Lanping A. Sung ◽  
Shu Chien
Keyword(s):  
Lectin Binding ◽  
Freeze Substitution ◽  
Electrostatic Charge ◽  
Geometric Parameters ◽  
Comparative Approach ◽  
Chemical Fixation ◽  
Cell Surfaces ◽  
Minimal Cell ◽  
Membrane Stiffness ◽  
Cell Contour

RBC agglutination by lectins represents an interactive balance between the attractive (bridging) force due to lectin binding on cell surfaces and disaggregating forces, such as membrane stiffness and electrostatic charge repulsion (1). During agglutination, critical geometric parameters of cell contour and intercellular distance reflect the magnitude of these interactive forces and the size of the bridging macromolecule (2). Valid ultrastructural measurements of these geometric parameters from agglutinated RBC's require preservation with minimal cell distortion. As chemical fixation may adversely influence RBC geometric properties (3), we used chemical fixation and cryofixation (rapid freezing followed by freeze-substitution) as a comparative approach to examine these parameters from RBC agglutinated with Ulex I lectin.

Freeze substitution fixation of fungal reproductive structures and spores

1989 ◽  
Vol 47 ◽  
pp. 990-991
Author(s):  
C. W. Mims ◽  
E. A. Richardson
Keyword(s):  
Plant Pathogens ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Dialysis Membrane ◽  
Razor Blade ◽  
Thin Sections ◽  
Reproductive Structures ◽  
Dry Down ◽  
Diamond Knife

The advantages of freeze substitution fixation over conventional chemical fixation for preservation of ultrastructural details in fungi have been discussed by various authors. As most ascomycetes, basidiomycetes and deuteromycetes do not fix well using conventional chemical fixation protocols, freeze substitution has attracted the attention of many individuals interested in fungal ultrastructure. Thus far most workers using this technique on fungi have concentrated on thin walled somatic hyphae. However, in our laboratory we have experimented with the use of freeze substitution on a variety of fungal reproductive structures and spores with promising results.Here we present data on freeze substituted samples of sporangia of the zygomycete Umbellopsis vinacea, basidia of Exobasidium camelliae var. gracilis, developing teliospores of the smut Sporisorium sorghi, germinating teliospores of the rust Gymnosporangium clavipes, germinating conidia of the deuteromycete Cercosporidium personatum, and developing ascospores of Ascodesmis nigricans.Spores of G. clavipes and C. personatum were deposited on moist pieces of sterile dialysis membrane where they hydrated and germinated. Asci of A. nigricans developed on pieces of dialysis membrane lying on nutrient agar plates. U. vinacea was cultured on small pieces of agar-coated wire. In the plant pathogens E. camelliae var. gracilis and S. sorghi, a razor blade was used to remove smal1 pieces of infected host issue. All samples were plunged directly into liquid propane and processed for study according to Hoch.l Samples on dialysis membrane were flat embedded. Serial thin sections were cut using a diamond knife, collected on slot grids, and allowed to dry down onto Formvar coated aluminum racks. Sections were post stained with uranyl acetate and lead citrate.

Immunocytochemistry after fast freeze fixation-freeze substitution: Advantages and limits

1990 ◽  
Vol 48 (3) ◽  
pp. 42-43
Author(s):  
Marie-Thérèse Nicolas
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Acrylic Resin ◽  
Expected Improvement ◽  
List Type ◽  
Chemical Fixation ◽  
Freeze Fixation ◽  
Liquid Chemical ◽  
Luciferase Enzyme ◽  
Lower Background

An alternative to aqueous chemical fixation consists in immobilizing physically the specimen by freezing it as fast as possible without using any cryoprotectant. This Fast Freeze Fixation (FFF) followed by Freeze Substitution (FS) avoids osmotic artefacts due to the slow penetration of liquid chemical fixative. Associated with Immuno-Gold labeling (IGS), FFF-FS allows a more precise localization of antigens.Using the bioluminescent bacteria Vibrio harveyi, a comparison of IGS with an antibody directed against its luciferase (enzyme of the luminescent reaction) has been done after liquid chemical fixation versus FFFFS. This later technique, beside an expected improvement of the ultrastructure always shows a better preservation of antigenicity and a lower background. In the case of FFF-FS technique (Figure 3):–labeling in acrylic resin (LRWhite) is 2 to 4 fold more intense than in epoxy resin (Epon),–but the ultrastructure is always better in Epon.–but the ultrastructure is always better in Epon.–The addition of fixatives in the substitution medium, results in a decrease of labeling which is more important in the case of a mixture of fixatives than with osmium tetroxide alone; with one exception: the substitution with glutaraldehyde which produces a dramatic increase in the density of the labeling but also, at the same time, a swelling of the cells of about 30%.

scholarly journals Immunolocalization of type III collagen in human articular cartilage prepared by high-pressure cryofixation, freeze-substitution, and low-temperature embedding.

1995 ◽  
Vol 43 (4) ◽  
pp. 421-427 ◽  
Author(s):  
R D Young ◽  
P A Lawrence ◽  
V C Duance ◽  
T Aigner ◽  
P Monaghan
Keyword(s):  
High Pressure ◽  
Articular Cartilage ◽  
Polyclonal Antibodies ◽  
Freeze Substitution ◽  
Immunogold Electron Microscopy ◽  
Type Iii Collagen ◽  
Human Articular Cartilage ◽  
Chemical Fixation ◽  
Osteoarthritic Cartilage ◽  
Type Iii

We localized Type III collagen by immunogold electron microscopy in resin sections of intact normal and osteoarthritic human articular cartilage. Comparisons of antibody staining between tissue prepared by high-pressure cryofixation and freeze-substitution without fixatives and that exposed to conventional mild chemical fixation with paraformaldehyde showed that dedicated cryotechniques yielded superior preservation of epitopes that are modified by chemical fixation, and simultaneously provided good ultrastructural preservation. Type III collagen was detected with two polyclonal antibodies, one against the triple-helical domain of the molecule and a second against the more antigenic, globular amino pro-peptide domain, which in this collagen is retained in the extracellular matrix after secretion. Positive labeling was seen in association with the major interstitial fibrils, suggesting co-polymerization of Types III and II collagen in cartilage. Type III collagen could not be detected in aldehyde-fixed normal cartilage. In fixed osteoarthritic cartilage, Type III was detectable only when the antibody to the amino pro-peptide was employed. In contrast, high-pressure cryofixation and freeze-substitution preserved epitopes for both antibodies, permitting immunodetection of Type III collagen in normal and osteoarthritic cartilage. Cryotechniques offer exciting possibilities for significantly improving the immunolocalization of collagens and other fixative-sensitive antigens in situ.

Cryo-SEM and TEM of High Pressure Frozen Cells - Some Technical Contributions

2001 ◽  
Vol 7 (S2) ◽  
pp. 728-729
Author(s):  
Paul Walther
Keyword(s):  
High Pressure ◽  
Physiological State ◽  
Electron Microscopic Observation ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Electron Microscopic ◽  
Biological Specimen ◽  
Freeze Fracturing

Imaging of fast frozen samples is the most direct approach for electron microscopy of biological specimen in a defined physiological state. It prevents chemical fixation and drying artifacts. High pressure freezing allows for ice-crystal-free cryo-fixation of tissue pieces up to a thickness of 200 urn and a diameter of 2 mm without prefixation. Such a frozen disc, however, is not directly amenable to electron microscopic observation: The structures of interest have to be made amenable to the electron beam, and the structures of interest must produce enough contrast to be recognized in the electron microscope. This can be achieved by freeze fracturing, cryo-sectioning or freeze substitution.The figures show high pressure frozen bakers yeast saccharomyces cerevisiae in the cryo-SEM (Figures 1 and 2) and after freeze substitution in the TEM (Figure 3). For high pressure freezing either a Bal-Tec HPM 010 (Princ. of Liechtenstein; Figures 1 and 2), or a Wohlwend HPF (Wohlwend GmbH, Sennwald, Switzerland; Figure 3) were used.

A plunge-freezing method for imaging mammalian tissue culture cells

1991 ◽  
Vol 49 ◽  
pp. 58-59
Author(s):  
E.T. O'Toole ◽  
J.R. McIntosh
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Freezing Method ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Humidity Chamber ◽  
Cellular Components ◽  
Coated Carbon

Ultrarapid freezing of tissue culture cells followed by freeze substitution has been used a method for the optimum fixation of cytoskeletal components that are often sensitive to routine chemical fixation. This is due to the fact that freezing methods such as plunge freezing, result in the almost instantaneous fixation of all cellular components without alteration of the cell's morphology. In addition, we have found that the plunge freezing method is useful for obtaining thin frozen cells for direct cryoimaging. Here we describe how the plunge freezing method can be applied both for freeze substitution analysis and for direct cryoimaging of frozen tissue culture cells.PTK, cells were grown to confluence on formvar coated, carbon stabilized gold grids. Prior to freezing, the grids were blotted in a 37°C, high humidity chamber so that a minimum of culture medium remained on the grid. This blotting step was critical to obtain a sample thin enough for optimum cryopreservation and subsequent cryoimaging.

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