Study of vitrified, unstained frozen tissue sections by cryoimmunoelectron microscopy

1991 ◽  
Vol 100 (1) ◽  
pp. 227-236
Author(s):  
I. Sabanay ◽  
T. Arad ◽  
S. Weiner ◽  
B. Geiger
Keyword(s):  
Heavy Metal ◽  
High Resolution ◽  
High Contrast ◽  
Frozen Sections ◽  
Microscope Examination ◽  
Tissue Sections ◽  
Novel Approach ◽  
Cytoplasmic Filaments ◽  
Frozen Tissue ◽  
Ultrathin Frozen Sections

We describe the development and application of a novel approach to high-resolution ultrastructural analysis of cells and tissues. It is based on the preparation of ultrathin frozen sections of fixed tissues, rinsing of the sections, followed by their embedding on the grid in a layer of vitrified ice, and direct observation with a cryoelectron microscope. Examination of smooth muscle, kidney and heart tissues showed that although no heavy metal staining was used, high-contrast images are obtained. Fine details of cytoplasmic filaments and organelles, membranes and membrane-associated structures, as well as connective-tissue elements are all visible. The new method is suitable for immunolabeling, including high resolution localization of specific molecules within the cytoplasm.

Freezing without Ice Crystal Damage: Semithin and Ultrathin Frozen Sections of Ethanol-Infiltrated Tissue for Microscopy, with Applications to Immunocytochemistry

1995 ◽  
Vol 1 (5) ◽  
pp. 217-230
Author(s):  
A. Kent Christensen ◽  
Terry B. Lowry
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light And Electron Microscopy ◽  
Ice Crystal ◽  
Freezing Damage ◽  
Frozen Sections ◽  
Glass Slides ◽  
Frozen Tissue ◽  
Crystal Damage ◽  
Ultrathin Frozen Sections

Ethanol (ethyl alcohol) has long been a standard reagent used in preparing tissues for light and electron microscopy. After fixation, tissues are usually dehydrated with ethanol before being embedded in paraffin or plastic. In this study we show that the ethanol-infiltrated tissue can be frozen and sectioned directly without embedding. When tissue impregnated with ethanol is cooled below about −117°C with liquid nitrogen, the ethanol solidifies without appreciable crystallization. The frozen tissue can then be sectioned in a commercial cryoultramicrotome that is set at −155 to −170°C to produce semithin frozen sections (0.5 to 3 μm thick) for light microscopy or ultrathin frozen sections (50 to 100 nm thick) for electron microscopy. Sections are picked up and mounted on glass slides or EM grids by means that are in current use for ice ultrathin frozen sectioning. Because there is no apparent freezing damage, the morphology in these ethanol frozen sections of unembedded tissue appears generally quite good, often resembling that obtained by conventional EM techniques. Examples are provided that illustrate the use of this material for immunocytochemistry at the light and electron microscope levels.

scholarly journals DETECTION OF DNA BY TRITIATED ACTINOMYCIN D ON ULTRATHIN FROZEN SECTIONS

1972 ◽  
Vol 53 (3) ◽  
pp. 798-808 ◽  
Author(s):  
Roch Bernier ◽  
Roberto Iglesias ◽  
René Simard
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Liver Tissue ◽  
Actinomycin D ◽  
Frozen Sections ◽  
Chromosomal Proteins ◽  
The Future ◽  
Complete Series ◽  
Ultrathin Frozen Sections ◽  
Cellular Components

Ultrathin frozen sections of fresh liver tissue were floated on actinomycin D-3H. Quantitative high resolution radioautography was performed to determine the value of the method for detection of DNA by electron microscopy. A complete series of control experiments involving various treatments of frozen sections with enzymes (pronase, DNase) and 0.1 N HCl were also carried out to determine the specificity of the labeling. The results indicate the value of the method for detection of DNA directly on ultrathin frozen sections. Short treatments with pronase followed by DNase reduce the labeling to zero, whereas removal of chromosomal proteins with HCl increases the amount of radioactivity in the nucleus considerably. The results are discussed in view of the future applications opened by ultracryotomy, since radioautographic detection of various macromolecules and cellular components by labeled compound with specific affinities will now be possible.

Mucin Stain on Frozen Sections

1999 ◽  
Vol 123 (5) ◽  
pp. 378-380
Author(s):  
Suchetha Soans ◽  
Lorenzo M. Galindo ◽  
Fernando U. Garcia
Keyword(s):  
Colonic Mucosa ◽  
Frozen Section ◽  
Staining Technique ◽  
Turnaround Time ◽  
Staining Procedure ◽  
Normal Colonic Mucosa ◽  
Frozen Sections ◽  
Tissue Sections ◽  
Paget Disease ◽  
Frozen Tissue

Abstract Context.—The importance of frozen-section diagnoses in the practice of pathology cannot be overemphasized. In some cases, the use of a mucin stain can greatly aid in the diagnosis. Since few methods for mucin staining have been described that can be used in the frozen-section setting, we developed one such staining procedure for mucin. Objective.—To develop a rapid mucicarmine staining technique to be used on frozen sections that does not significantly delay overall turnaround time. Design.—A standard mucicarmine staining technique was modified by using a concentrated mucicarmine stain and a microwave oven, to reduce the total staining time to 3 minutes or less. Frozen tissue from normal colonic mucosa was used as a control, and skin from extramammary Paget disease for evaluation of margins was used as a case. Results.—The rapid mucicarmine stain successfully demonstrated the presence of mucin on frozen-tissue sections. Mucin stained deep rose, and the connective tissue stained green. Conclusion.—This rapid and simple mucin staining technique can be used on frozen sections with no significant effect on the overall turnaround time, thereby aiding rapid diagnosis on frozen sections.

scholarly journals Mixed glycosidase pretreatment reduces nonspecific binding of antibodies to frozen tissue sections.

1985 ◽  
Vol 33 (7) ◽  
pp. 695-698 ◽  
Author(s):  
L P Andrews ◽  
R K Clark ◽  
I Damjanov
Keyword(s):  
Monoclonal Antibody ◽  
Renal Tissue ◽  
Frozen Sections ◽  
Tissue Sections ◽  
Mouse Tissues ◽  
Background Staining ◽  
Frozen Tissue ◽  
Nonspecific Staining ◽  
Immunohistochemical Studies ◽  
Monoclonal Antibody Binding

Indirect immunohistochemical studies of frozen mouse tissues with mouse monoclonal antibodies yield, in general, suboptimal results primarily because of indiscriminate binding of secondary antibody to all mouse immunoglobulins, i.e., to the monoclonal reagent and to endogenous immunoglobulin nonspecifically trapped in the tissue. To reduce this nonspecific staining, frozen sections of mouse kidney were treated enzymatically. Optimal results were obtained following a 2 hr treatment with 20 mg/ml of mixed glycosidases (MG). This treatment reduced the nonspecific background staining of the interstitial spaces and blood vessels, but did not affect the reactivity of structurally bound immunoglobulin G (IgG) in the glomeruli or alter the reactivity of mouse renal tissue to the monoclonal antibody that recognizes an oligosaccharide antigenic determinant (SSEA-1). Eluates from enzyme-treated frozen tissue sections contained normally immunoreactive IgG in the form of dimers. These data indicate that MG treatment of frozen sections could be safely used to reduce the content of nonstructurally bound immunoglobulins in frozen tissues and thus improve the visualization of specific monoclonal antibody binding.

scholarly journals Immunoelectronmicroscopy of neural antigens on ultrathin frozen sections.

1986 ◽  
Vol 34 (4) ◽  
pp. 491-500 ◽  
Author(s):  
M R Celio ◽  
G A Keller ◽  
F E Bloom
Keyword(s):  
High Resolution ◽  
Organic Solvents ◽  
Ultrastructural Level ◽  
Frozen Sections ◽  
Neuronal Function ◽  
Enzyme Marker ◽  
Ultrathin Frozen Sections

We have utilized immunocryoultramicrotomy to detect synapsin, somatostatin, parvalbumin, and tubulin at the ultrastructural level. Immunocryoultramicrotomy combines informative identification of morphology with accurate immunolabeling. Moreover, since no detergents or organic solvents are used to enable antibody penetration, and since no enzyme marker diffusion occurs, localization of the antigens should be more accurate. Accordingly, it was possible to localize precisely all four antigens within a well-preserved structure. Application of this method has important advantages for high-resolution localization of molecules relevant to neuronal function.

Immunoferritin localization of intracellular antigens in positively stained frozen sections

1978 ◽  
Vol 36 (2) ◽  
pp. 168-169
Author(s):  
K. T. Tokuyasu
Keyword(s):  
Surface Tension ◽  
Water Surface ◽  
Thin Layers ◽  
The Other ◽  
Positive Staining ◽  
Frozen Sections ◽  
The Past ◽  
Ultrathin Frozen Sections ◽  
Definition Of

During the past investigations of immunoferritin localization of intracellular antigens in ultrathin frozen sections, we found that the degree of negative staining required to delineate u1trastructural details was often too dense for the recognition of ferritin particles. The quality of positive staining of ultrathin frozen sections, on the other hand, has generally been far inferior to that attainable in conventional plastic embedded sections, particularly in the definition of membranes. As we discussed before, a main cause of this difficulty seemed to be the vulnerability of frozen sections to the damaging effects of air-water surface tension at the time of drying of the sections.Indeed, we found that the quality of positive staining is greatly improved when positively stained frozen sections are protected against the effects of surface tension by embedding them in thin layers of mechanically stable materials at the time of drying (unpublished).

Localization of pancreatic enzymes in ultrathin frozen sections stained with metal labeled antibody

1978 ◽  
Vol 36 (2) ◽  
pp. 90-91
Author(s):  
Kenjiro Yasuda
Keyword(s):  
Fluorescent Antibody ◽  
Pancreatic Enzymes ◽  
Tissue Preparation ◽  
Zymogen Granules ◽  
Frozen Sections ◽  
En Bloc ◽  
Antibody Method ◽  
Ultrathin Frozen Sections ◽  
Fluorescent Antibody Method

Localization of amylase,chymotrypsinogen and trypsinogen in pancreas was demonstrated by Yasuda and Coons (1966), by using fluorescent antibody method. These enzymes were naturally found in the zymogen granules. Among them, amylase showed a diffuse localization around the nucleus, in addition to the zymogen granules. Using ferritin antibody method, scattered ferritin granules were also found around the Golgi area (Yasuda et al.,1967). The recent advance in the tissue preparation enables the antigen to be localized in the ultrathin frozen sections, by applying the labeled antibodies onto the sections instead of staining the tissue en bloc.The present study deals with the comparison of the localization of amylase and lipase demonstrated by applying the bismuth-labeled, peroxidase-labeled and ferritin-labeled antibody methods on the ultrathin frozen sections of pancreas, and on the blocks of the same tissue.

The Application of Ultracryotomy to Immunocytochemistry

1973 ◽  
Vol 31 ◽  
pp. 704-709
Author(s):  
R. G. Painter ◽  
K. T. Tokuyasu ◽  
S. J. Singer
Keyword(s):  
Frozen Sections ◽  
Protein Antigens ◽  
Carbon Coated ◽  
Antibody Conjugates ◽  
Ultrathin Frozen Sections

A technique for localizing intracellular antigens with immunoferritin conjugates directly on ultrathin frozen sections of glutaraldehyde-fixed tissues has been developed. This method overcomes some of the limitations of previously described procedures, since it avoids drastic fixation, dehydration and embedding procedures which could denature many protein antigens.Briefly cells or tissues were fixed with glutaraldehyde (0.5 to 2% for 1 hr), and ultrathin frozen sections were cut and mounted on grids covered with carbon-coated Formvar film by the procedure described previously. Such sections were stained with ferritin-antibody conjugates by methods described elsewhere.

Ultrathin frozen sections of yeast without aldehyde prefixation

1978 ◽  
Vol 36 (2) ◽  
pp. 58-59
Author(s):  
K. J. Böhm ◽  
a. E. Unger
Keyword(s):  
Aqueous Solutions ◽  
Biological Material ◽  
Yeast Cells ◽  
Frozen Sections ◽  
Good Preservation ◽  
Ultrathin Frozen Sections

During the last years it was shown that also by means of cryo-ultra-microtomy a good preservation of substructural details of biological material was possible. However the specimen generally was prefixed in these cases with aldehydes.Preparing ultrathin frozen sections of chemically non-prefixed material commonly was linked up to considerable technical and manual expense and the results were not always satisfying. Furthermore, it seems to be impossible to carry out cytochemical investigations by means of treating sections of unfixed biological material with aqueous solutions.We therefore tried to overcome these difficulties by preparing yeast cells (S. cerevisiae) in the following manner:

Recent Progress In High-Resolution Shadowing For Biological Transmission Electron Microscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 510-511 ◽  
Author(s):  
Heinz Gross ◽  
Katarina Krusche ◽  
Peter Tittmann
Keyword(s):  
Heavy Metal ◽  
High Resolution ◽  
Freeze Drying ◽  
Atmospheric Conditions ◽  
Vacuum Equipment ◽  
Transmission Electron ◽  
Gas Atmosphere ◽  
Gas Molecules ◽  
Residual Gas ◽  
Backing Layer

Freeze-drying followed by heavy metal shadowing is a long established and straight forward approach to routinely study the structure of dehydrated macromolecules. Very thin specimens such as isolated membranes or single macromolecules are directly adsorbed on C-coated grids. After rapid freezing the grids are transferred into a suitable vacuum equipment for freeze-drying and heavy metal shadowing.To improve the resolution power of shadowing films we introduced shadowing at very low specimen temperature (−250°C). To routinely do that without the danger of contamination we developed in collaboration with Balzers an UHV (p≤10-9 mbar) machine (BAF500K, Fig.2). It should be mentioned here that at −250°C the specimen surface acts as effective cryopump for practically all impinging residual gas molecules from the residual gas atmosphere.Common high resolution shadowing films (Pt/C, Ta/W) have to be protected from alterations due to air contact by a relatively thick C-backing layer, when transferred via atmospheric conditions into the TEM. Such an additional C-coat contributes disturbingly to the contrast at high resolution.

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