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 Formaldehyde as a Fixative for Light and Electron Microscopy

2000 ◽  
Vol 8 (5) ◽  
pp. 30-31
Author(s):  
Freida L. Carson
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Parallel Study ◽  
Electron Microscopic ◽  
Dual Purpose ◽  
Ultrastructural Morphology ◽  
The World ◽  
Selection Of

Since Blum discovered its hardening properties in 1893, formaldehyde has become the most widely used fixative in the world for specimens to be examined by light microscopy. However, since most commercial preparations of formaldehyde contain methanol, a protein precipitant, formaldehyde has been considered an unsatisfactory fixative for tissues to be examined by electron microscopy. In 1973, Carson et al., described a parallel study comparing the electron microscopic results of fixation with paraformaidehyde vs. formaldehyde. They found that there was no difference in the preservation of ultrastructural morphology provided that the buffer systems were identical. In 1976, McDowell and Trump described a fixative combining commercial formaldehyde and glutaraldehyde (4CF-1G). Both of these fixatives are dual purpose fixatives and preclude the selection of tissue for electron microscopy prior to fixation. They can both be prepared in large quantities and used for routine surgical specimens. The fixative containing formaldehyde alone does not need to be refrigerated and is stable for months; whereas, the formaldehyde-glutaraldehyde mixture should be refrigerated.

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.

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.

Ruthenium red-osmium bridging with TCH: new technique to stain biological specimens for light and TEM and to coat them for SEM

1988 ◽  
Vol 46 ◽  
pp. 20-21
Author(s):  
B. Giammara ◽  
J. Hanker
Keyword(s):  
Electron Microscopy ◽  
Microscopic Study ◽  
Osmium Tetroxide ◽  
Light And Electron Microscopy ◽  
Ruthenium Red ◽  
Surface Coat ◽  
Repeated Application ◽  
Electron Microscopic ◽  
Sputter Coating ◽  
Selection Of

The demonstration and coating of glycomacromolecular surface coat components of biological specimens (1) with ruthenium red (RR, Fig. 1) is improved by treating with osmium tetroxide (2) probably due to its attachment to glycolipids. Since 1966 studies have shown how bridging osmium to osmium with thiocarbohydrazide (TCH, Fig. 2) can result in improvement in contrast of biological specimens (3,4) for light and electron microscopy. Since 1973 this bridging procedure has widely been applied (5,6) to obtain a conductive coating for biological specimens for SEM eliminating the need for sputter coating. Improvement in conductance of uncoated specimens for EM has also been obtained (6) by bridging osmium with p-phenylenediamine hydrochloride (PPD). The improvement in conductance of RR coated biological specimens for SEM by OsO4 treatment without TCH (2) required repeated application of the reagent solutions and did not result in sufficient staining of the glycomacromolecules and glycolipids for the light microscopic selection of areas for electron microscopic study.

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.

scholarly journals Visualization of Identified GFP-expressing Cells by Light and Electron Microscopy

2003 ◽  
Vol 51 (3) ◽  
pp. 271-274 ◽  
Author(s):  
Katherine Luby-Phelps ◽  
Gang Ning ◽  
Joseph Fogerty ◽  
Joseph C. Besharse
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Immunogold Labeling ◽  
Living Tissue ◽  
Gfp Expression ◽  
Fixed Tissue ◽  
Lr White ◽  
Thin Sectioning ◽  
Gfp Fluorescence ◽  
Semithin Sections

We have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1-μm sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning.

Contrast Enhancement of Fresh Frozen-Dried Tissue for Light and Electron Microscopy

1977 ◽  
Vol 35 ◽  
pp. 548-549
Author(s):  
Patrick W. Bankston ◽  
Louis Terracio
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Biological Tissues ◽  
Ice Crystal ◽  
Fixed Tissue ◽  
X Ray ◽  
Fresh Frozen ◽  
Electron Microscopes ◽  
Specific Reactions ◽  
Cellular Components

Recent improvements in the technique of freeze-drying now routinely provide biological tissues with morphological preservation ultrastructurally comparable to conventionally-fixed tissue. Using this technique, tissue is frozen on a metal surface, dried in vacuo, vapor fixed with OSO4 and directly embedded in polymers suitable for sectioning for light or electron microscopy. Tissue processed in this manner can be used for such specialized applications as x-ray microanalysis and radioautography of soluble substances. Further, it is possible to eliminate OSO4 before embedding making frozen-dried tissue useful for investigation of cellular components by cytochemical reactions performed on sectioned material. Many of these procedures require that the tissue be free of OSO4 initially but that OSO4 be present for final viewing. In addition, frozen-dried tissue requires preliminary viewing in the light and electron microscopes to evaluate the extent of ice crystal damage before use for specific reactions. However, it is difficult to stain unfixed plastic embedded tissue for light microscopy and equally difficult to provide adequate contrast to these for electron microscopy.

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 THE PRESERVATION OF THE FINE STRUCTURE OF CRYOSTAT-SECTIONED TISSUE WITH DIMETHYLSULFOXIDE FOR COMBINED LIGHT AND ELECTRON MICROSCOPY

1968 ◽  
Vol 16 (1) ◽  
pp. 40-48 ◽  
Author(s):  
DANIEL ZAGURY ◽  
PAT G. MODEL ◽  
GEORGE D. PAPPAS
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Electron Microscope ◽  
Light And Electron Microscopy ◽  
Freezing And Thawing ◽  
Electron Microscopes ◽  
Flat Embedding ◽  
Cryostat Sections ◽  
Sectioned Tissue ◽  
Selection Of

A method which allows the observation of identical tissue components with both the light and electron microscopes is presented. The technique is based on the preparation of cryostat sections of organs with dimethylsulfoxide. This agent appears to prevent the damage to fine structure caused by freezing and thawing. The use of plastic sheets permits flat embedding of organ sections in Epon. Examination of the embedded sections, stained or unstained, in the light microscope allows the exact selection of material to be studied further in the electron microscope.

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.

Sign in / Sign up

Export Citation Format

Share Document