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

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.

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Cryofixation of Plant Material for Ultrastructura1 and Analytical Studies

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600003469 ◽

1980 ◽

Vol 38 ◽

pp. 796-797

Author(s):

Patrick Echlin ◽

Clifford Lai ◽

Albert Saubermann ◽

Thomas Hayes ◽

Helen Skaer ◽

...

Keyword(s):

Plant Material ◽

Bulk Material ◽

Water Soluble ◽

Ice Crystal ◽

Frozen Sections ◽

Animal Tissues ◽

Water Soluble Polymers ◽

Soluble Polymers ◽

Analytical Studies ◽

Crystal Damage

Studies (1—4) have shown that high-molecular weight, water soluble polymers such as polyvinylpyrrolidone (PVP) and hydroxyethyl starch (HES) are useful in reducing ice-crystal damage in rapidly frozen samples of plant and animal tissues as judged by their appearance in freeze-etch replicas, frozen sections, and frozen fracture faces of bulk material. We have extended these studies to the preparation of material for correlative ultrastructural and analytical studies where it is important both to retain diffusible elements in their natural position in cell as well as preserving adequate cell morphology.

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Immunoferritin localization of intracellular antigens in positively stained frozen sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008170x ◽

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).

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081310 ◽

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.

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The Application of Ultracryotomy to Immunocytochemistry

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073969 ◽

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.

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Ultrathin frozen sections of yeast without aldehyde prefixation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081152 ◽

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:

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