A comparison of neuropeptide immunocytochemistry in fluid-fixed and freeze-dried brains

1981 ◽  
Vol 220 (2) ◽  
pp. 223-230 ◽  
Author(s):  
C. J. Clayton ◽  
T. H. McNeill ◽  
J. R. Sladek
Keyword(s):  
Neuropeptide Immunocytochemistry ◽  
Freeze Dried

scholarly journals Video-enhanced technique for detecting neurophysin, enkephalin, and somatostatin immunoreactivity in ultrathin sections of cat median eminence.

1986 ◽  
Vol 34 (11) ◽  
pp. 1405-1415 ◽  
Author(s):  
H D Coulter ◽  
R P Elde
Keyword(s):  
Median Eminence ◽  
Freeze Drying ◽  
Video Camera ◽  
Neuropeptide Immunocytochemistry ◽  
Serial Sections ◽  
External Zone ◽  
Freeze Dried ◽  
Ultrathin Sections ◽  
Low Levels ◽  
Somatostatin Immunoreactivity

A freeze-drying technique using epoxy-embedded ultrathin serial sections permits critical comparisons of neuropeptides in small fibers and varicosities of the nervous system by video-enhanced, light microscopic immunofluorescence. The desirability of the method was documented by data showing: retention of radioimmunoassayable somatostatin in freeze-substituted blocks of tissue as compared to its loss in tissue dehydrated in an alcohol series; feasibility of OsO4 vapor fixation of freeze-dried tissue and compatibility with neuropeptide immunocytochemistry, and utility of a silicon-intensified-tube video camera for recording low levels of fluorescence from ultrathin sections. Ultrathin serial sections, 150 nm thick, from the inner zone of freeze-dried median eminence of the cat revealed three populations of axons containing various combinations of neurophysin immunoreactivity and enkephalin immunoreactivity. Some elements contained neurophysin immunoreactivity alone, some contained both neurophysin immunoreactivity and enkephalin immunoreactivity, and a few elements contained enkephalin immunoreactivity alone. The adjacent external zone of the median eminence contained immunoreactivity for all three substances, but the structures in this region were too small to permit demonstration of coexistence in 150 nm thick sections.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

Scanning and Transmission Microscopy of Nematocyst Batteries in Epitheliomuscular Cells of Hydra

1971 ◽  
Vol 29 ◽  
pp. 410-411 ◽  
Author(s):  
Jane A. Westfall ◽  
S. Yamataka ◽  
Paul D. Enos
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Osmium Tetroxide ◽  
External Surface ◽  
Three Dimensional ◽  
Surface Structures ◽  
Transmission Microscopy ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Electron

Scanning electron microscopy (SEM) provides three dimensional details of external surface structures and supplements ultrastructural information provided by transmission electron microscopy (TEM). Animals composed of watery jellylike tissues such as hydras and other coelenterates have not been considered suitable for SEM studies because of the difficulty in preserving such organisms in a normal state. This study demonstrates 1) the successful use of SEM on such tissue, and 2) the unique arrangement of batteries of nematocysts within large epitheliomuscular cells on tentacles of Hydra littoralis.Whole specimens of Hydra were prepared for SEM (Figs. 1 and 2) by the fix, freeze-dry, coat technique of Small and Màrszalek. The specimens were fixed in osmium tetroxide and mercuric chloride, freeze-dried in vacuo on a prechilled 1 Kg brass block, and coated with gold-palladium. Tissues for TEM (Figs. 3 and 4) were fixed in glutaraldehyde followed by osmium tetroxide. Scanning micrographs were taken on a Cambridge Stereoscan Mark II A microscope at 10 KV and transmission micrographs were taken on an RCA EMU 3G microscope (Fig. 3) or on a Hitachi HU 11B microscope (Fig. 4).

Characterization of microtubules by dark-field STEM and replication

1991 ◽  
Vol 49 ◽  
pp. 152-153
Author(s):  
S.B. Andrews ◽  
R.D. Leapman ◽  
P.E. Gallant ◽  
T.S. Reese
Keyword(s):  
Protein Interactions ◽  
Low Dose ◽  
Unit Length ◽  
Dark Field ◽  
Carbon Films ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Freeze Dried ◽  
Protein Motors ◽  
Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

The Effects of Drying Techniques on Clay-Rich Soil Texture

1974 ◽  
Vol 32 ◽  
pp. 466-467
Author(s):  
T. G. Naymik
Keyword(s):  
Critical Point ◽  
Liquid Nitrogen ◽  
Liquid Nitrogen Temperature ◽  
Drying Methods ◽  
Air Drying ◽  
Freeze Dried ◽  
Critical Point Drying ◽  
Set Up ◽  
The Relationship ◽  
Quartz Grains

Three techniques were incorporated for drying clay-rich specimens: air-drying, freeze-drying and critical point drying. In air-drying, the specimens were set out for several days to dry or were placed in an oven (80°F) for several hours. The freeze-dried specimens were frozen by immersion in liquid nitrogen or in isopentane at near liquid nitrogen temperature and then were immediately placed in the freeze-dry vacuum chamber. The critical point specimens were molded in agar immediately after sampling. When the agar had set up the dehydration series, water-alcohol-amyl acetate-CO2 was carried out. The objectives were to compare the fabric plasmas (clays and precipitates), fabricskeletons (quartz grains) and the relationship between them for each drying technique. The three drying methods are not only applicable to the study of treated soils, but can be incorporated into all SEM clay soil studies.

Oncornavirus assembly at the cell surface revealed in replicas of freeze-dried cells

1978 ◽  
Vol 36 (2) ◽  
pp. 354-355
Author(s):  
Anthony Demsey ◽  
Christopher W. Stackpole
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Viral Origin ◽  
Intact Cells ◽  
Structural Formation ◽  
Virus Core ◽  
Freeze Dried ◽  
Cacodylate Buffer ◽  
Surface Replica ◽  
Leukemia Viruses

The murine leukemia viruses are type-C oncornaviruses, and their release from the host cell involves a “budding” process in which the newly-forming, RNA-containing virus core becomes enveloped by modified cell surface membrane. Previous studies revealed that the released virions possess a dense array of 10 nm globular projections (“knobs”) on this envelope surface, and that these knobs contain a 70, 000 MW glycoprotein (gp70) of viral origin. Taking advantage of this distinctive structural formation, we have developed a procedure for freeze-drying and replication of intact cells which reveals surface detail superior to other surface replica techniques, and sufficient to detect even early stages of virus budding by localized aggregation of these knobs on the cell surface.Briefly, cells growing in monolayer are seeded onto round glass coverslips 10-12 mm in diameter. After a period of growth, cells are fixed in situ for one hour, usually with 1% OsO4 in 0. 1 M cacodylate buffer, and rinsed in distilled water.

Conformation of eukaryotic ribosomal RNAs

1983 ◽  
Vol 41 ◽  
pp. 592-593
Author(s):  
M. Boublik ◽  
G. Thornton ◽  
G. Oostergetel ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Molecular Weight ◽  
Mass Measurement ◽  
Carbon Film ◽  
Structural Complexity ◽  
Scanning Transmission Electron Microscope ◽  
Electron Microscopic ◽  
Pure Nitrogen ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Partially Unfolded

Understanding the structural complexity of ribosomes and their role in protein synthesis requires knowledge of the conformation of their components - rRNAs and proteins. Application of dedicated scanning transmission electron microscope (STEM), electrical discharge of the support carbon film in an atmosphere of pure nitrogen, and determination of the molecular weight of individual rRNAs enabled us to obtain high resolution electron microscopic images of unstained freeze-dried rRNA molecules from BHK cells in a form suitable for evaluation of their 3-D structure. Preliminary values for the molecular weight of 28S RNA from the large and 18S RNA from the small ribosomal subunits as obtained by mass measurement were 1.84 x 106 and 0.97 x 106, respectively. Conformation of rRNAs consists, in general, of alternating segments of intramolecular hairpin stems and single stranded loops in a proportion which depends on their ionic environment, the Mg++ concentration in particular. Molecules of 28S RNA (Fig. 1) and 18S RNA (not shown) obtained by freeze-drying from a solution of 60 mM NH+4 acetate and 2 mM Mg++ acetate, pH 7, appear as partially unfolded coils with compact cores suggesting a high degree of ordered secondary structure.

Scanning Electron Microscopy-cuticular Lesions on the Roundworm Ascaris Suum

1970 ◽  
Vol 28 ◽  
pp. 114-115
Author(s):  
P. A. Madden ◽  
W. R. Anderson
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Freeze Drying ◽  
Room Temperature ◽  
Secondary Electron ◽  
Distilled Water ◽  
Freeze Dried ◽  
Silver Paint ◽  
To Come ◽  
Scanning Electron

The intestinal roundworm of swine is pinkish in color and about the diameter of a lead pencil. Adult worms, taken from parasitized swine, frequently were observed with macroscopic lesions on their cuticule. Those possessing such lesions were rinsed in distilled water, and cylindrical segments of the affected areas were removed. Some of the segments were fixed in buffered formalin before freeze-drying; others were freeze-dried immediately. Initially, specimens were quenched in liquid freon followed by immersion in liquid nitrogen. They were then placed in ampuoles in a freezer at −45C and sublimated by vacuum until dry. After the specimens appeared dry, the freezer was allowed to come to room temperature slowly while the vacuum was maintained. The dried specimens were attached to metal pegs with conductive silver paint and placed in a vacuum evaporator on a rotating tilting stage. They were then coated by evaporating an alloy of 20% palladium and 80% gold to a thickness of approximately 300 A°. The specimens were examined by secondary electron emmission in a scanning electron microscope.

Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

1988 ◽  
Vol 46 ◽  
pp. 176-177
Author(s):  
J.S. Wall ◽  
V. Maridiyan ◽  
S. Tumminia ◽  
J. Hairifeld ◽  
M. Boublik
Keyword(s):  
Ionic Strength ◽  
Three Dimensional ◽  
Conformational Transitions ◽  
Dark Field ◽  
Dimensional Structure ◽  
Ribosomal Rnas ◽  
Field Mode ◽  
Freeze Dried ◽  
High Resolution Images ◽  
Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

Quantitative Diameter Measurements of Chromatin and TMV Fibers in the Freeze-Dried State

1988 ◽  
Vol 46 ◽  
pp. 170-171
Author(s):  
B. D. Athey ◽  
A. L. Stout ◽  
M. F. Smith ◽  
J. P. Langmore
Keyword(s):  
Reliable Method ◽  
Fiber Diameter ◽  
Quantitative Agreement ◽  
One Dimension ◽  
Fiber Axis ◽  
Two Dimensional ◽  
Fiber Density ◽  
Variable Diameter ◽  
Freeze Dried ◽  
Expected Values

Although there is general agreement that Inactive chromosome fibers consist of helically packed nucleosomes, the pattern of packing is still undetermined. Only one of the proposed models, the crossed-linker model, predicts a variable diameter dependent on the length of DNA between nucleosomes. Measurements of the fiber diameter of negatively-stained and frozen- hydrated- chromatin from Thyone sperm (87bp linker) and Necturus erythrocytes (48bp linker) have been previously reported from this laboratory. We now introduce a more reliable method of measuring the diameters of electron images of fibrous objects. The procedure uses a modified version of the computer program TOTAL, which takes a two-dimensional projection of the fiber density (represented by the micrograph itself) and projects it down the fiber axis onto one dimension. We illustrate this method using high contrast, in-focus STEM images of TMV and chromatin from Thyone and Necturus. The measured diameters are in quantitative agreement with the expected values for the crossed-linker model for chromatin structure

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