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.

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.

Electron microscopic analysis of objects in light microscopic sections

1978 ◽  
Vol 36 (2) ◽  
pp. 80-81
Author(s):  
Arvid B. Maunsbach
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Microscopic Analysis ◽  
Electron Microscopic Level ◽  
Semithin Section ◽  
Cover Glass ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Glass Slides ◽  
Ultrathin Sections

Structural studies in experimental biology or in pathology are frequently extended from the light to the electron microscopic level. This is often done by cutting both semithin (about 1 μm) and thin sections from the same tissue block after embedding for electron microscopy. However, in many studies it would be of great value to analyse the same structure both by light and electron microscopy, i.e. to be able to study by electron microscopy an object which is first detected by light microscopy in a semithin section. To achieve this, a method has been developed by which ultrathin sections are cut directly from the semithin section containing the object of interest.Semithin sections, about 1 μ in thickness, are cut from Epon or Vestopal embedded tissue. The sections are placed on ordinary glass slides and stained with toluidine blue. The sections are studied in the light microscope without a cover glass or mounted in water.

Cell migration from the chick olfactory placode: a light and electron microscopic study

Development ◽  
1982 ◽  
Vol 69 (1) ◽  
pp. 47-59
Author(s):  
A. S. Mendoza ◽  
W. Breipohl ◽  
F. Miragall
Keyword(s):  
Electron Microscopy ◽  
Cell Migration ◽  
Light And Electron Microscopy ◽  
Olfactory Mucosa ◽  
Electron Microscopic ◽  
Olfactory Placode ◽  
The Third ◽  
Epitheloid Cells ◽  
Migration Of Cells ◽  
Number Of Cells

The diflferentiation of the olfactory placode in the chick has been studied using light and electron microscopy. Special attention was paid to the appearance of neuronal cells within the placodal ectodermal thickening, the migration of cells out of this tissue and the appearance of the first fila olfactoria in the differentiating olfactory mucosa. Between the third and fifth day of incubation a large number of cells is observed leaving the base of the invaginating olfactory placode, often in contact with thin axon bundles. These cells are characterized by a well-developed Golgi apparatus, a considerable number of mitochondria and dense-core vesicles. The morphology of these migrating cells resembles that of cells observed near the basement membrane within the developing olfactory epithelium and is clearly different from the mesenchymal cells which are filled with polyribosomes. At the sixth day of incubation thick axon bundles can be observed within the epithelium and the underlying lamina propria. The possible fate of the migrated epitheloid cells is discussed.

Early embryology af the marsupials Isoodon macrourus and Perameles nasuta

1976 ◽  
Vol 24 (3) ◽  
pp. 361 ◽  
Author(s):  
AG Lyne ◽  
DE Hollis
Keyword(s):  
Electron Microscopy ◽  
Outer Surface ◽  
Zona Pellucida ◽  
Light And Electron Microscopy ◽  
Blastocyst Stage ◽  
Embryonic Cells ◽  
Crystalloid Inclusions ◽  
Early Mouse ◽  
Number Of Cells ◽  
Rabbit Embryos

Twelve embryos, ranging from a four-celled stage to late unilaminar blastocysts, were obtained from the bandicoots I. macrourus and P. nasuta and examined by light and electron microscopy. These early stages covered at least one-quarter of the 12.5-day gestation period. The three non-cellular egg membranes characteristic of marsupials (zona pellucida, mucoid coat and shell membrane) were present, although the zona was sometimes absent or discontinuous in the intermediate and late unilaminar blastocysts examined. At the four-celled stage the blastomeres were close to the zona, but they had lost contact with each other, probably due to the extrusion of yolk, a phenomenon which has been described in other marsupials. The embryo did not increase in diameter until it was composed of at least 75 contiguous cells, which were in contact with the zona. In several of the larger blastocysts the protoderm cells had lost contact with the zona. Subsequently, the number of cells increased considerably and they were flattened against the egg membranes to form the late unilaminar blastocyst stage. Electron microscopy of the protoderm cells revealed the presence of numerous microvilli, particularly on the outer surface, and a range of other structures as great as those found in eutherian mammals. Remnants of spindle bridges were common in one 75-celled embryo. The yolk material in the blastocoele was also composed of a variety of structures, including small crystalloid inclusions composed of hexagonal units about 8-10 nm in diameter. Similar crystalloids have been described in the cells of early mouse and rabbit embryos and in egg and embryonic cells of various amphibians.

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.

The “rediscovery” of polyethylene glycol, and its use as an embedding matrix

1984 ◽  
Vol 42 ◽  
pp. 28-31
Author(s):  
J. J. Wolosewick
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Epoxy Resins ◽  
Room Temperature ◽  
Light And Electron Microscopy ◽  
Polyethylene Glycols ◽  
Frozen Sections ◽  
Solid Polymers ◽  
Standard Procedures ◽  
Peg 4000

Polyethylene glycols are liquid or solid polymers of the general formula H(OCH2CH2)nOH, where n is greater than or equal to 4. They are readily miscible in water and in a variety of solvents and are nontoxic. The higher molecular weight polymers (e.g., PEG 4000, 6000) are brittle and melt at 55°-65°C, while the lower molecular weight polymers are soft gummy solids or liquids. These polymers have been used as embedding matrices for light and electron microscopy since the 1940's, although their use has been overshadowed by the now standard embedments (e.g., paraffin and epoxy resins).The procedures for embedding in PEG have been described in detail. Briefly, specimens are fixed according to standard procedures, washed in an appropriate buffer, dehydrated either in PEG-H2O solutions, or in ethyl alcohol. For embedding, the specimens are transferred to capsules filled with the pure polymer and solidified at room temperature, or by rapid cooling in liquid nitrogen. The blocks are mounted onto suitable stubs (e.g., Epon blanks), trimmed and sectioned “dry” on glass or diamond knives. The sections are mounted onto polylysine-coated slides or Forrmvar-coated grids in the same manner as are frozen sections.

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 Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Pieter Baatsen ◽  
Sergio Gabarre ◽  
Katlijn Vints ◽  
Rosanne Wouters ◽  
Dorien Vandael ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Fluorescent Proteins ◽  
Fluorescent Microscopy ◽  
Light And Electron Microscopy ◽  
High Resolution Electron Microscopy ◽  
Fluorescence Signal ◽  
Microscopy Imaging ◽  
Tissue Samples ◽  
Rapid Acquisition

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

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