Acanthamoeba castellanii: ultrastructure of trophozoites using fast freeze-fixation followed by freeze-substitution

2001 ◽  
Vol 50 (5) ◽  
pp. 423-427 ◽  
Author(s):  
A. Gonzalez-Robles
Keyword(s):  
Acanthamoeba Castellanii ◽  
Freeze Substitution ◽  
Freeze Fixation

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%.

Fast freeze-fixation/freeze-substitution reveals the secretory membranes of the gastric parietal cell as a network of helically coiled tubule. A new model for parietal cell transformation

1995 ◽  
Vol 108 (3) ◽  
pp. 1127-1141 ◽  
Author(s):  
J.M. Pettitt ◽  
D.C. Humphris ◽  
S.P. Barrett ◽  
B.H. Toh ◽  
I.R. van Driel ◽  
...  
Keyword(s):  
Parietal Cell ◽  
Apical Membrane ◽  
Cell Transformation ◽  
Freeze Substitution ◽  
Membrane System ◽  
Morphological Transformation ◽  
Tubular Membrane ◽  
Unstimulated Cell ◽  
Helical Coils ◽  
Freeze Fixation

The parietal cell of the gastric mucosa undergoes rapid morphological transformation when it is stimulated to produce hydrochloric acid. In chemically fixed cells, this process is seen as a reduction in number of cytoplasmic ‘tubulovesicles’ as the apical surface of the cell progressively invaginates to increase the secretory surface area. It is widely believed that the tubulovesicles represent stored secretory membrane in the cytoplasm of the unstimulated cell, which is incorporated into the apical membrane upon stimulation, because they share H+,K+-ATPase activity with the apical membrane. However, fusion of tubulovesicles with the apical membrane concomitant with parietal cell activation has never been convincingly demonstrated. We have used fast freeze-fixation and freeze-substitution to study stages of morphological transformation in these cells. Tubulovesicles were not seen in the cytoplasm of any of our cryoprepared cells. Instead, the cytoplasm of the unstimulated cell contained numerous and densely packed helical coils of tubule, each having an axial core of cytoplasm. The helical coils were linked together by connecting tubules, lengths of relatively straight tubule. Lengths of straight connecting tubule also extended from coils lying adjacent to the apical and canalicular surfaces and ended at the apical and canaliculus membranes. Immunogold labelling with alpha- and beta-subunit-specific antibodies showed that the gastric H+,K+-ATPase was localized to the membranes of this tubular system, which therefore represented the configuration of the secretory membrane in the cytoplasm of the unstimulated parietal cell. Stimulation of the cells with histamine and isobutylmethylxanthine lead to modification of the tubular membrane system, correlated with progressive invagination of the apical membrane. The volume of the tubule lumen increased and, as this occurred, the tight spiral twist of the helical coils was lost, indicating that tubule distension was accounted for by partial unwinding. This exposed the cores of cytoplasm in the axes of the coils as rod-shaped elements of a three-dimensional reticulum, resembling a series of microvilli in random thin sections. Conversely, treatment with the H2 antagonist cimetidine caused severe contraction of the tubular membrane system and intracellular canaliculi. Our results indicate that tubulovesicles are an artifact of chemical fixation; consequently, they cannot have a role in parietal cell transformation. From our findings we propose an alternative model for morphological transformation in the parietal cell. This model predicts cytoskeleton-mediated control over expansion and contraction of the tubular membrane network revealed by cryopreparation. The model is compatible with the localization of cytoskeletal components in these cells.

High-pressure freeze fixation reveals novel features during ontogenesis of the vegetative cell in Ledebouria pollen: an ultrastructural and cytochemical study

1995 ◽  
Vol 73 (1-2) ◽  
pp. 1-10 ◽  
Author(s):  
Michael W. Hess
Keyword(s):  
High Pressure ◽  
Vegetative Cell ◽  
Spatial Organization ◽  
Spatial Relationship ◽  
Freeze Substitution ◽  
Structural Features ◽  
Protein Bodies ◽  
Lipid Digestion ◽  
Cytochemical Study ◽  
Freeze Fixation

The ultrastructure of the vegetative cell in the pollen of Ledebouria socialis Roth (Hyacinthaceae) was investigated from microspore mitosis to anthesis. As a result of the good preservation quality achieved with high-pressure freeze fixation and freeze substitution, novel structural features were observed. Extensive endomembrane compartments emerging at the onset of lipid and starch mobilization, were identified as protein bodies by using video-enhanced contrast light microscopy. Thus, proteins, apart from starch and lipids, represent a third class of important intermediary storage substances in developing pollen. The close spatial relationship between protein bodies, endoplasmic reticulum (ER), and storage lipids suggest that protein bodies and ER contribute to lipid digestion. Immediately prior to anthesis the protein bodies become transformed into unspecialized vacuoles as a result of the gradual dissolution of their contents; the formation of the protein bodies remains still to be elucidated. The ER proliferates extensively during pollen ontogenesis, thereby changing its ultrastructure and spatial organization. Microfilaments were detected during all developmental stages, in particular microtubule-associated single microfilaments. The microfilaments are likely to be composed of actin as shown by immunogold labeling.Key words: angiosperm pollen, freeze substitution, protein bodies, microfilaments, Hyacinthaceae.

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.

Sign in / Sign up

Export Citation Format

Share Document