scholarly journals Immunogold labeling of luciferase in the luminous bacterium Vibrio harveyi after fast-freeze fixation and different freeze-substitution and embedding procedures.

1989 ◽  
Vol 37 (5) ◽  
pp. 663-674 ◽  
Author(s):  
M T Nicolas ◽  
J M Bassot ◽  
G Nicolas
Keyword(s):  
Vibrio Harveyi ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Immunogold Labeling ◽  
Chemical Fixation ◽  
Luminous Bacterium ◽  
Immunogold Staining ◽  
Bioluminescent Bacterium ◽  
Lr White ◽  
Freeze Fixation

We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.

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 Ultrastructural localization of HTLV-I gag proteins p19 and p24 by single and double immunogold labeling.

1991 ◽  
Vol 39 (2) ◽  
pp. 185-191 ◽  
Author(s):  
G Stransky ◽  
S Gay
Keyword(s):  
Osmium Tetroxide ◽  
Decisive Role ◽  
Ultrastructural Localization ◽  
Immunogold Labeling ◽  
Gag Proteins ◽  
Lr White ◽  
Positive Immunostaining ◽  
Infected Cells ◽  
Lowicryl K4m

We developed a post-embedding immunogold labeling procedure for the ultrastructural localization of the HTLV-I gag proteins p19 and p24 by the use of monoclonal antibodies (MAb). Both antigens were shown to withstand fixation with 1% glutaraldehyde. In addition, p19 antigenicity was found not to be affected by post-fixation with 1% osmium tetroxide. The choice of resin played a decisive role in the retention of antigenicity. P19 was preserved in Lowicryl K4M as well as in LR White, whereas p24 was preserved only in Lowicryl. Both p19 and p24 were found to be localized on the HTLV-I virions themselves, whereas no positive immunostaining could be observed on the infected cells. In Lowicryl-embedded samples, in which both antigens had been preserved, a double immunogold labeling procedure was performed that allowed the co-localization of p19 and p24 on the same section. In osmicated LR White-embedded samples the quality of ultrastructural preservation of HTLV-I virions was found to be comparable to results obtained with the traditional glutaraldehyde-osmium tetroxide-epoxy resin processing.

scholarly journals Characterization of the bioluminescent organelles in Gonyaulax polyedra (dinoflagellates) after fast-freeze fixation and antiluciferase immunogold staining.

1987 ◽  
Vol 105 (2) ◽  
pp. 723-735 ◽  
Author(s):  
M T Nicolas ◽  
G Nicolas ◽  
C H Johnson ◽  
J M Bassot ◽  
J W Hastings
Keyword(s):  
Freeze Substitution ◽  
Vacuolar Membrane ◽  
Immunogold Staining ◽  
Gonyaulax Polyedra ◽  
Light Emitting ◽  
Dense Bodies ◽  
Structural Relationships ◽  
Freeze Fixation ◽  
Membrane Bound

To characterize the microsources of bioluminescent activity in the dinoflagellate Gonyaulax polyedra, an immunogold labeling method using a polyclonal antiluciferase was combined with fast-freeze fixation and freeze substitution. The quality of the preservation and the specificity of the labeling were greatly improved compared to earlier results with chemical fixation. Two organelles were specifically labeled: cytoplasmic dense bodies with a finely vermiculate texture, and mature trichocysts, labeled in the space between the shaft and the membrane. The available evidence indicates that the dense bodies are the light-emitting microsources observed in vivo. The dense bodies appear to originate in the Golgi area as cytoplasmic densifications and, while migrating peripherally, come into contact with the vacuolar membrane. Mature organelles protrude and hang like drops in the vacuolar space, linked by narrow necks to the cytoplasm. These structural relationships, not previously apparent with glutaraldehyde fixation, suggest how bioluminescent flashes can be elicited by a proton influx from a triggering action potential propagated along the vacuolar membrane. Similar dense bodies were labeled in the active particulate biochemical fraction (the scintillons), where they were completely membrane bound, as expected if their necks were broken and resealed during extraction. The significance of the trichocyst reactivity remains enigmatic. Both organelles were labeled with affinity-purified antibody, which makes it unlikely that the trichocyst labeling is due to a second antibody of different specificity. But trichocysts are not bioluminescent; the cross-reacting material could be luciferase present in this compartment for some other reason, or a different protein carrying similar antigenic epitopes.

scholarly journals In Situ Localization of Two Fibrillar Collagens in Two Compact Connective Tissues by Immunoelectron Microscopy After Cryotechnical Processing

1997 ◽  
Vol 45 (1) ◽  
pp. 119-128 ◽  
Author(s):  
G. Nicolas ◽  
F. Gaill ◽  
L. Zylberberg
Keyword(s):  
Osmium Tetroxide ◽  
Type I Collagen ◽  
Freeze Substitution ◽  
Immunogold Electron Microscopy ◽  
Type I ◽  
Fish Scale ◽  
Electron Microscopic ◽  
Lr White ◽  
Fibrillar Collagens ◽  
Freeze Fixation

Two fibrillar collagens, the worm cuticular collagen and the vertebrate Type I fish scale collagen, both organized in a compact tissue, were localized by immunogold electron microscopy in resin sections after freeze-fixation and freeze-substitution. Identification of these two fibrillar collagens failed with the-use of postembedding labeling after conventional electron microscopic processing. Positive labeling of the Type I collagen was observed in sections of fish scales freeze-fixed by either slam-freezing or high-pressure freezing, freeze-substituted in acetone with or without osmium tetroxide, and embedded in LR White. The worm cuticular collagen was detected in sections of cuticle that were freeze-fixed, freeze-substituted (necessarily with osmium tetroxide added to acetone), and embedded in either LR White or Epon. It was also detected in specimens pre-fixed by aldehydes before freeze-fixation. The Type I fish scale collagen appears to be more sensitive than the fibrillar cuticular collagen of worms to the procedures employed for postembedding immunoelectron microcopy. Our results have shown that freeze-fixation and freeze-substitution preserved the antigenicity of the fibrillar collagens organized in a compact three-dimensional network, whereas immunolabeling failed after conventional electron microscopic procedures. These cryostabilization techniques appear to be of value to improve the immunolocalization of collagens.

Ultrastructural localization of neuropeptides in the rat brain

1978 ◽  
Vol 36 (2) ◽  
pp. 612-613
Author(s):  
F.W. Van Leeuwen
Keyword(s):  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Serial Sections ◽  
Microscopical Level ◽  
Electron Microscopical Level ◽  
Enzyme Method ◽  
Perfusion Fixation ◽  
Electron Microscopical ◽  
Unlabeled Antibody ◽  
Peroxidase Conjugate

In order to obtain specific and optimal ultrastructural localization of vasopressin and oxytocin in the hypothalamo-neurohypophyseal system of the rat, 2 staining procedures and several tissue treatments were evaluated using neurohypophyseal tissue. It appeared from these studies that post-embedding staining with the unlabeled antibody enzyme method developed by Sternberger allows greater dilution of the first antibody (anti-vasopressin, 1:4800) than the indirect procedure (1:320) using a peroxidase conjugate as second antibody. Immersion fixation with 4% formalin during 24 hours gave better results (general ultrastructure, immunoreactivity) than those obtained by perfusion fixation with 2.5% glutaraldehyde-1% paraformaldehyde or freeze substitution.Since no reliable specificity tests were performed at the electron microscopical level, tests were developed for antibodies against both vasopressin and oxytocin. For anti-vasopressin plasma neural lobes of homozygous Brattleboro rats, that are lacking vasopressin by a genet- ical defect, were used. For antibodies against both hormones serial sections were used that were alternately incubated with the antibodies.

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.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

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.

scholarly journals An artificial test substrate for evaluating electron microscopic immunocytochemical labeling reactions.

1987 ◽  
Vol 35 (8) ◽  
pp. 909-916 ◽  
Author(s):  
G D Gagne ◽  
M F Miller
Keyword(s):  
Electron Microscope ◽  
Hepatitis B ◽  
Horseradish Peroxidase ◽  
Polyclonal Antibodies ◽  
Hepatitis B Surface Antigen ◽  
Immunogold Labeling ◽  
Artificial Substrate ◽  
Chemical Fixation ◽  
Electron Microscopic ◽  
Immunocytochemical Studies

We describe an artificial substrate system for optimization of labeling parameters in electron microscope immunocytochemical studies. The system involves use of blocks of glutaraldehyde-polymerized BSA into which a desired antigen is incorporated by a simple soaking procedure. The resulting antigen-impregnated artificial substrate can then be fixed and embedded identically to a piece of tissue. The BSA substrate can also be dried and then sectioned for immunolabeling with or without chemical fixation and without exposing the antigen to dehydrating agents and embedding resins. The effects of various fixation and embedding procedures can thus be evaluated separately. Other parameters affecting immunocytochemical labeling, such as antibody and conjugate concentration, can also be evaluated. We used this system, along with immunogold labeling, to determine quantitatively the optimal fixation and embedding conditions for labeling of hepatitis B surface antigen (HbsAg), human IgG, and horseradish peroxidase. Using unfixed and unembedded HBsAg, we were able to detect antigen concentrations below 20 micrograms/ml. We have shown that it is not possible to label HBsAg within resin-embedded cells using conventional aldehyde fixation protocols and polyclonal antibodies.

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