Ultrathin frozen sections for Electron Microscopy: Some personal recollections

1992 ◽  
Vol 50 (2) ◽  
pp. 1080-1081
Author(s):  
A. Kent Christensen
Keyword(s):  
Electron Microscopy ◽  
Dimethyl Sulfoxide ◽  
Cell Biology ◽  
Steroid Hormone ◽  
Uranyl Acetate ◽  
Frozen Sections ◽  
Fixed Tissue ◽  
Hormone Synthesis ◽  
Ultrathin Frozen Sections

In the mid-1960s, while on the faculty of the Anatomy Department at Stanford, I was particularly interested in the cell biology of steroid-secreting cells. I had studied the ultrastructure of these cells, and was anxious to trace the pathways of steroid hormone synthesis and of the secretion from the cell. An invitation to speak at an international steroid congress in Milan, Italy, in May 1966, afforded me an opportunity to travel in Europe before the meeting started. During that trip I had a very enjoyable visit with Dr. Wilhelm Bernhard, in the Paris suburb Villejuif. He had developed means of cutting ultrathin frozen sections (UFS) of fixed tissue on a Sorvall MT-1 ultramicrotome maintained in a freezer at about −35°C. As the sections were cut, they floated off on a solution of dimethyl sulfoxide and water, from which they were picked up on EM grids, treated for cytochemistry, stained with uranyl acetate, and then viewed by EM.

scholarly journals ULTRATHIN FROZEN SECTIONS

1967 ◽  
Vol 34 (3) ◽  
pp. 757-771 ◽  
Author(s):  
W. Bernhard ◽  
Elizabeth H. Leduc
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Phosphotungstic Acid ◽  
Cultured Cells ◽  
Uranyl Acetate ◽  
Distilled Water ◽  
Frozen Sections ◽  
Simple Method ◽  
Ultrathin Frozen Sections

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25–4% glutaraldehyde for 1–4 hr, are washed overnight in buffer at 3°C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70°C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35°C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.

scholarly journals Visualization of longitudinally-oriented intermediate filaments in frozen sections of chicken cardiac muscle by a new staining method.

1983 ◽  
Vol 97 (2) ◽  
pp. 562-565 ◽  
Author(s):  
K T Tokuyasu
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Intermediate Filaments ◽  
Ethyl Cellulose ◽  
Staining Method ◽  
Uranyl Acetate ◽  
Frozen Sections ◽  
Cell Biol ◽  
Ultrathin Frozen Sections

When ultrathin frozen sections of chicken cardiac muscle were osmicated, dehydrated in ethanol, embedded in ethyl cellulose, and stained with acidic uranyl acetate, filaments of 10-12 nm width were visualized in wide interfibrillar spaces. Immunostaining of the frozen sections for desmin resulted in exclusive labeling of such filaments. These observations indicated that longitudinally oriented networks of intermediate filaments were present in the interfibrillar spaces, in addition to the transversely oriented networks that surround myofibrils at the level of Z band. As in skeletal muscle (Tokuyasu, K. T., A. H. Dutton, and S. J. Singer, 1983, J. Cell Biol. 97:1727-1735), desmin in chicken cardiac muscle is believed to be largely, if not entirely, in the form of intermediate filaments.

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.

Simultaneous observations of immunolabeled frozen sections in lm and em

1978 ◽  
Vol 36 (2) ◽  
pp. 164-165
Author(s):  
K. T. Tokuyasu ◽  
J. Slot ◽  
S. J. Singer
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Microscopy ◽  
Light And Electron Microscopy ◽  
Frozen Sections ◽  
Cover Glass ◽  
Rat Pancreas ◽  
Light Microscopic Observation ◽  
Rabbit Igg ◽  
Ultrathin Frozen Sections ◽  
Number Of Cells

Immunofluorescent microscopy is more suitable for the analysis of a large number of cells, often greater in the sensitivity for the detection of antigens, and more readily applicable for the identification of multiple antigens than immunoe1ectron microscopy. For combining these features of fluorescent microscopy with the superior resolution of electron microscopy, we attempted to observe the same immunolabeled ultrathin frozen sections with both light and electron microscopy.Ultrathin frozen sections of rat pancreas fixed in a mixture of 2% formaldehyde and 0.2% g1utaraldehyde for 1 hr at 4°C were first immunostained with rabbit anti-rat amylase antibodies, then very lightly with ferritin-goat anti-rabbit IgG conjugates and heavily with rhodamine-goat anti-rabbit IgG conjugates. For light microscopic observation, grids were suspended underneath the cover glass with a very thin layer of 50-90% glycerol and the cover glass was separated from the slide glass by a spacer to avoid the contact of the grid with the slide glass. After the light microscope observation, the grids were floated on 0.1 M phosphate buffer by dissolving glycerol into the buffer and processed for electron microscopy.

Adsorption Staining Methd For Ultrathin Frozen Sections

1980 ◽  
Vol 38 ◽  
pp. 760-763
Author(s):  
K. T. Tokuyasu
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Negative Staining ◽  
Positive Staining ◽  
Frozen Sections ◽  
Transmission Electron ◽  
Ultrathin Sections ◽  
Immunocytochemical Studies ◽  
Ultrathin Frozen Sections

The successful routine use of cryoultramicrotomy to examine ultrathin sections by transmission electron microscopy requires the application of suitable staining to delineate the ultrastructure. While negative staining is quite effective for certain purposes1, 2, positive staining is more appropriate for immunocytochemical studies because it does not obscure the immunolabels.

scholarly journals Method for epitope selection of antisera for immunohistology.

1989 ◽  
Vol 37 (2) ◽  
pp. 273-276 ◽  
Author(s):  
D Wedlich
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Antigen Detection ◽  
Frozen Sections ◽  
Fixed Tissue ◽  
Nitrocellulose Filter ◽  
Epitope Selection ◽  
Immunohistological Analysis ◽  
Polyclonal Serum ◽  
Selection Of

Polyclonal anti-laminin serum was affinity-purified on paraformaldehyde-fixed laminin on a nitrocellulose filter. The purified antibodies were tested for their specificity in immunohistological stainings on frozen sections of paraformaldehyde-fixed tissue. As compared to the initial polyclonal serum, the purified antibodies increased the specificity of antigen detection, since all background caused by nonspecific reactions was eliminated. This technique promises to be very useful for immunohistological analysis using light and electron microscopy.

scholarly journals IMPROVED TECHNIQUES FOR THE PREPARATION OF ULTRATHIN FROZEN SECTIONS

1971 ◽  
Vol 49 (3) ◽  
pp. 731-746 ◽  
Author(s):  
W. Bernhard ◽  
Annie Viron
Keyword(s):  
Electron Microscopy ◽  
Biological Tissues ◽  
Frozen Sections ◽  
Large Molecules ◽  
Recent Introduction ◽  
Morphological Studies ◽  
Plastic Embedding ◽  
Ultrathin Frozen Sections

Ultrathin frozen sections of biological tissues for electron microscopy provide certain advantages in cytochemical studies in which the penetration of cells by large molecules is necessary and in morphological studies of cellular constituents which are dissolved by the reagents employed in routine plastic embedding. The recent introduction of several types of commercially available cryo-ultramicrotomes makes it possible for many laboratories to employ this valuable tool. This paper summarizes recent improvements in the methods developed in this laboratory for preparing ultrathin frozen sections and reviews some of the inherent problems involved in their use. These procedures may serve as a baseline for other investigators who can then modify or adapt them for their specific purposes.

scholarly journals LOCALIZATION OF ACID PHOSPHATASE ACTIVITY IN HEPATIC LYSOSOMES BY MEANS OF ELECTRON MICROSCOPY

1961 ◽  
Vol 9 (4) ◽  
pp. 773-784 ◽  
Author(s):  
E. Essner ◽  
Alex B. Novikoff
Keyword(s):  
Electron Microscopy ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Human Liver ◽  
Osmium Tetroxide ◽  
Frozen Sections ◽  
Fixed Tissue ◽  
Thin Sections ◽  
Lipofuscin Granules

Samples of liver from untreated rats, from rats infused with unconjugated bilirubin, and from biopsies of human liver were fixed overnight in cold formol-calcium. Frozen sections were stained for acid phosphatase activity by the Gomori lead-glycerophosphate procedure. Small blocks of fixed tissue were also incubated in this medium. These were then treated briefly with osmium tetroxide, dehydrated, and embedded in methacrylate. Thin sections were studied by electron microscopy. The sites of reaction product of acid phosphatase activity as visualized in electron micrographs are consistent with those seen in frozen sections studied by light microscopy. They indicate that the pericanalicular bodies of parenchymatous cells, the large spherical bodies of Kupffer cells, the microbodies appearing after bilirubin infusion and lipofuscin granules belong to the class of cytoplasmic organelles called lysosomes by de Duve.

scholarly journals Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

1976 ◽  
Vol 24 (5) ◽  
pp. 621-629 ◽  
Author(s):  
N Shepard ◽  
N Mitchell
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Toluidine Blue ◽  
Light And Electron Microscopy ◽  
Uranyl Acetate ◽  
Collagen Fibrils ◽  
Epiphyseal Cartilage ◽  
Fixation Method ◽  
Fixed Tissue

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.

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