scholarly journals Negative-staining autoradiography: a new technique for ultracryotomy utilizing an interposed film.

1986 ◽  
Vol 34 (8) ◽  
pp. 1085-1094 ◽  
Author(s):  
Y Futaesaku ◽  
V Mizuhira
Keyword(s):  
Retention Rate ◽  
Cryostat Section ◽  
Ultrastructural Localization ◽  
Negative Staining ◽  
Alkaline Solutions ◽  
Gelatin Layer ◽  
Ultrastructural Damage ◽  
A New Technique ◽  
Cryostat Sections ◽  
Silver Grains

A new radiocytochemical technique is reported for ultrastructural localization of diffusible substances, using negatively stained ultra-cryostat sections. A sheet of film interposed between the cryostat section and the emulsion layer has rendered negative-staining autoradiography (NSA) practical. The rationale of NSA is that the film completely shields the section from all moisture-producing autoradiographic processes, so that phosphotungstic acid (PTA) can stain the section either before or after autoradiography (ARG), without the possibility of ultrastructural damage by alkaline solutions, interference between PTA and photoprocessing compounds, and superimposed images of a gelatin layer stained with PTA. As a model to demonstrate the newly developed procedure of NSA, rat brains were labeled with [125I]-triiodothyronine, fixed with tannic fixative, immersed in a cryoprotectant, frozen in liquefied propane, and cryostat sectioned. The resulting higher yield of radioactivity (85%) on the section was confirmed by a radiation counter. The retention rate was approximately 20% greater than that of conventional sections. Developed silver grains were found on synaptic vesicles and mitochondria in the polymorphic layer of the dentate gyrus. In this report we will also discuss the problems associated with cryostat sectioning of fresh tissues, the concept of ARG resolution, the distribution pattern of developed silver grains, and the possible applications of NSA.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCES IN THE MOUSE COLON WITH IRON-CONTAINING STAINS

1966 ◽  
Vol 30 (2) ◽  
pp. 299-315 ◽  
Author(s):  
Mary G. Wetzel ◽  
Bruce K. Wetzel ◽  
Samuel S. Spicer
Keyword(s):  
Ferric Chloride ◽  
Ultrastructural Localization ◽  
Cell Types ◽  
Capillary Endothelium ◽  
Colloidal Iron ◽  
Specific Staining ◽  
Mouse Colon ◽  
Specific Localization ◽  
Cryostat Sections ◽  
Acid Mucosubstances

Selective ultrastructural staining of acid mucosubstances in sites containing histochemically identifiable sulfo- and sialomucins has been obtained in fixed cryostat sections with both ferric chloride and colloidal iron solutions. The rectosigmoid region of mouse colon was fixed in glutaraldehyde, formalin, or phosphate-buffered osmium tetroxide, and 40 µ cryostat sections of this material were treated with 0.1 to 0.4% ferric chloride or with a solution of dialyzed ferric chloride, ammonia, and glycerin. Specific staining depended upon the pH of the iron-containing solutions, and the optimal value was found to be approximately 2.0. Specific localization of acid mucosubstances has been noted in intracellular sites, including globules within colonic goblet cells and "deep crypt" mucous cells, small vesicles of the superficial nongoblet epithelial cells, and Golgi lamellae within each of these cell types. Extracellular material, presumed to be acid mucosubstance, was found on the surface of the epithelial microvilli and on the lumenal surface of capillary endothelium. Similar material formed a reticular network surrounding stromal cells, collagen bundles, and various colonic connective tissue elements.

scholarly journals Autometallographic Silver Enhancement of Zinc Sulfide Crystals Created in Cryostat Sections from Human Brain Biopsies: A New Technique that Makes it Feasible to Demonstrate Zinc Ions in Tissue Sections from Biopsies and Early Autopsy Material

1997 ◽  
Vol 45 (11) ◽  
pp. 1503-1510 ◽  
Author(s):  
Gorm Danscher ◽  
S⊘ren Juhl ◽  
Meredin Stoltenberg ◽  
Bjarne Krunderup ◽  
Henrik D. Schr⊘der ◽  
...  
Keyword(s):  
Human Brain ◽  
Zinc Sulfide ◽  
Significant Loss ◽  
New Technique ◽  
Zinc Ions ◽  
Autopsy Material ◽  
Silver Enhancement ◽  
Crystal Lattices ◽  
A New Technique ◽  
Cryostat Sections

We present a new technique that allows zinc ions in synaptic and secretory vesicles of biopsy and early autopsy material (>2 hr post mortem) to be transformed to nanometer-sized zinc sulfide crystal lattices for subsequent autometallographic (AMG) development. Human brain biopsies, or other tissue samples containing zinc-enriched (ZEN) cells, are frozen in liquid nitrogen or by CO2 gas immediately after removal. The tissue blocks are cut in a cryostat and the sections placed on glass slides. The slides are transferred to an H2S exposure chamber placed in a −15C freezer. After 1–24 hr of gas exposure the sections are removed from the chamber, fixed while thawing, and dehydrated. The sections are then exposed to an AMG developer. AMG causes silver enhancement of zinc sulfide crystal lattices created in the tissues through the H2S exposure, making them visible. It is imperative that the tissues are frozen instantaneously after removal, because loosely bound or free zinc ions start leaving their vesicular compartment soon after death. The AMG technique can, despite inadequate fixation and damage to the tissue caused by freezing, also be used to trace zinc ions at ultrastructural levels, and it is demonstrated that zinc ions in the human neocortex are located in synaptic vesicles. In the few human biopsies analyzed thus far, the light microscopic pattern created by the silver-enhanced ZEN terminals resembles that seen in the neocortex of rat brain. The technique has been applied to cryostat sections from neocortex biopsies of five individuals undergoing brain surgery. Biopsies from three patients resulted in satisfactory AMG-stained sections. Rat brains removed and frozen immediately after decapitation constituted the material on which the present technique was developed. Such material results in an almost uniform high quality of staining, and we found that unexposed sections can be stored for at least 5 months at −80C without ensuing significant loss of AMG staining intensity. (J Histochem Cytochem 45:1503–1510, 1997)

scholarly journals THE MICROCRYSTALLINE STRUCTURE OF CELLULOSE IN CELL WALLS OF COTTON, RAMIE, AND JUTE FIBERS AS REVEALED BY NEGATIVE STAINING OF SECTIONS

1966 ◽  
Vol 29 (2) ◽  
pp. 181-197 ◽  
Author(s):  
A. N. J. Heyn
Keyword(s):  
Cell Walls ◽  
Spatial Arrangement ◽  
Negative Staining ◽  
X Ray Diffraction ◽  
Indirect Methods ◽  
X Ray ◽  
Jute Fibers ◽  
Ultrathin Sections ◽  
A New Technique ◽  
Microfibrillar Structure

With a new technique of negative staining of sections, it has been possible to observe directly, in ultrathin sections under the electron microscope, the original microcrystalline and microfibrillar structure of cellulose as it occurs in living cells. This method has advantages over the study of isolated fibers used so far by others, in that the original arrangement of microfibrils is better preserved, and their collapse into larger fibrillar units is prevented. With this method, the cell walls of ramie, jute, and cotton fibers have been studied. The size (diameter, 25 to 40 A) and the longitudinal periodicity observed in the single microfibrils and the orientation and spatial arrangement of the microcrystallite within the microfibrils are found to correspond with the latest models derived by others from data obtained by indirect methods such as X-ray diffraction. The microfibril size of about 35 A, found by measuring these structures in sections, agrees with the latest conclusions reached by others in recent work with isolated fibrils.

scholarly journals Ultrastructural Localization of Calcium in Ischemic Hippocampal Slices: The Influence of Adenosine and Theophylline

1992 ◽  
Vol 12 (3) ◽  
pp. 520-524 ◽  
Author(s):  
Ernö Dux ◽  
Peter Schubert ◽  
Georg W. Kreutzberg
Keyword(s):  
Morphological Changes ◽  
Hippocampal Slices ◽  
Ultrastructural Localization ◽  
Free Medium ◽  
Adenosine Receptor Antagonist ◽  
Ca1 Region ◽  
Calcium Sequestration ◽  
Ultrastructural Damage ◽  
Induced Changes ◽  
Neuroprotective Action

Calcium was localized ultrastructurally with the use of the modified oxalate-pyroantimonate reaction in the CA1 region of rat hippocampal slices. Ten-minute ischemia (incubation with anoxic and glucose-free medium) followed by 30 min reoxygenation resulted in mitochondrial calcium sequestration and ultrastructural damage. The addition of the adenosine receptor antagonist, theophylline, worsened the ischemia-induced morphological changes and particularly exaggerated the Ca2+ loading in the postsynaptic dendrites. In contrast, adenosine protected against ischemia-induced changes. The results suggest that adenosine exerts its neuroprotective action largely by maintaining intracellular calcium-homeostasis.

Differential Averaging of Single Molecule Images using Multivariate Statistical Classification

1982 ◽  
Vol 40 ◽  
pp. 706-709
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscope ◽  
Single Molecule ◽  
Negative Staining ◽  
New Technique ◽  
Statistical Classification ◽  
Multivariate Statistical ◽  
Local Averaging ◽  
Resolution Level ◽  
A New Technique ◽  
Range Of Variation

It has been shown that averaging of single molecules or molecular assemblies visualized in the electron microscope is capable of giving reproducible results at the resolution level allowed by negative staining. The resolution achieved for a given specimen is, however, dependent upon the range of variation that the molecule exhibits in the electron microscope (fig. 1). A new technique, differential or local averaging, promises to overcome this limitation in fully utilizing the information contained in the experimental images.

scholarly journals A NEW DIAZO REAGENT FOR SPECIFIC STAINING OF CONJUGATED BILIRUBIN IN TISSUE SECTIONS

1968 ◽  
Vol 16 (6) ◽  
pp. 419-427 ◽  
Author(s):  
V. J. DESMET ◽  
A.-M. BULLENS ◽  
J. DE GROOTE ◽  
K. P. M. HEIRWEGH
Keyword(s):  
Total Bilirubin ◽  
Diazonium Salt ◽  
Histochemical Demonstration ◽  
Specific Method ◽  
Bile Pigments ◽  
Specific Staining ◽  
Tissue Sections ◽  
A New Technique ◽  
Cryostat Sections ◽  
Conjugated Bilirubin

A new technique is presented for the specific histochemical demonstration of conjugated bilirubin in tissue sections, using the diazonium salt of ethylanthranilate. The specificity of this method was proved by using test materials and cholestatic tissue sections. There was no diffusion artifact under the prescribed working conditions. The method is applicable to fresh cryostat sections and frozen sections of cold, formol-calcium-fixed tissues. The method could not be satisfactorily applied for the staining of total bilirubin. The method using 2,4-dichloraniline remains the best available technique for the histochemical demonstration of total bilirubin, while the technique with ethylanthranilate proves to be the most specific method available for the demonstration of conjugated bile pigments.

scholarly journals A new method for the cytochemical demonstration of peroxidase for light, fluorescence and electron microscopy.

1976 ◽  
Vol 24 (1) ◽  
pp. 82-90 ◽  
Author(s):  
J M Papadimitriou ◽  
P Van Duijn ◽  
P Brederoo ◽  
J G Streefkerk
Keyword(s):  
Peroxidase Activity ◽  
Fluorescent Dyes ◽  
Green Light ◽  
Ultrastructural Localization ◽  
Complex Salt ◽  
Lead Sulphide ◽  
Enzyme Cytochemistry ◽  
Cytochemical Demonstration ◽  
A New Technique ◽  
Light Fluorescence

A new technique for the cytochemical demonstration of peroxidase is presented. Monomeric homovanillic acid is converted by H2O2 and peroxidase into its dimeric form, which is then precipitated as a complex salt of lead and rhodamine 6G or rhodamine B. The reaction product can be visualized by conversion to lead sulphide or viewed directly under the fluorescence microscope, since it emits a red fluorescence when excited with green light. The reaction is rapid and results in good localization at the cytologic level of peroxidase activity in granulocytes. The technique can be applied for the ultrastructural localization of enzymatic activity, but in its present form it does not match the localization sharpness of the diaminobenzidine method. This fluorescent cytochemical technique will also detect horseradish peroxidase activity and may provide a usefull probe in peroxidase immunohistochemistry. The principle of complexing metal salts with fluorescent dyes may find a more general application in enzyme cytochemistry.

Ultrastructural localization of carbonic anhydrase in mouse gastric parietal cells

1978 ◽  
Vol 36 (2) ◽  
pp. 532-533
Author(s):  
N. Sugai ◽  
S. Ito
Keyword(s):  
Carbonic Anhydrase ◽  
Picric Acid ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Parietal Cells ◽  
Thin Sections ◽  
Tissue Sections ◽  
Gastric Parietal Cells ◽  
Cryostat Sections ◽  
Precise Distribution

The histochemical localization of carbonic anhydrase in parietal cells has been described by a number of investigators and its presence in these cells at the ultrastructural level has been also reported. However the precise distribution of this enzyme is not clear and there are some questions regarding the validity of the histochemical reaction. In the present study, various modifications of the technique were explored and it was found that tissues fixed in buffered formaldehyde, glutaraldehyde with trinitrocresol or picric acid retained good reaction product localization of this enzyme. Cryostat sections of the fixed tissues were treated with solutions recommended by Hannson. Incubation times that were most favorable 5 to 10 min for light microscopy and 8 to 15 min for electron microscopy. For ultrastructural observations of thin sections, it was found to be important that the reacted tissue sections were post osmicated with 1% osmium tetroxide in 1. 5% potassium ferrocyanide or with aqueous 1% 0s04 for only 3 to 5 min.

A Method for the Ultrastructural Localization of Sucrase

1972 ◽  
Vol 30 ◽  
pp. 6-7
Author(s):  
V. Lorenzsonn ◽  
H. Agresti ◽  
W. A. Olsen
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Ultrastructural Localization ◽  
Negative Staining ◽  
Sucrase Activity ◽  
Alternative Method ◽  
Brush Borders ◽  
Surface Localization ◽  
Microvillus Membrane ◽  
Intestinal Sucrase

Several studies have suggested that intestinal sucrase is located in 60 Å particles of microvillus membrane seen with negative staining. This localization has been questioned, however, because of a disparity between the disappearance of particles and enzyme activity from brush borders during 20 min. papain digestions. Ferritin labeled antibody to sucrase has also been used to study surface localization of the enzyme. We have devised an alternative method to allow us to study intracellular localization as well for study of the increased sucrase activity in diabetic mucosa. We modified the technique of Keston for ultrastructural use by replacing the chromagen with 3,3'-diaminobenzidine tetrahydrochloride (DAB).

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF MINERAL MATTER IN BACTERIAL SPORES BY MICROINCINERATION

1964 ◽  
Vol 23 (1) ◽  
pp. 113-133 ◽  
Author(s):  
Richard S. Thomas
Keyword(s):  
Fine Structure ◽  
Electron Microscope ◽  
Middle Layer ◽  
Ultrastructural Localization ◽  
Mineral Elements ◽  
Dark Field ◽  
Mineral Matter ◽  
A New Technique ◽  
Definition Of ◽  
Excited Oxygen

The fine localization of mineral matter in spores of Bacillus megaterium and Bacillus cereus was studied by the technique of microincineration adapted for use with the electron microscope. The specimens, which included intact and thin-sectioned spores as well as shed spore coats, were burned either in the conventional way at high temperature or by a new technique using electrically excited oxygen at nearly room temperature. The ash residues were examined by bright field, dark field, and diffraction in the electron microscope and also with the phase contrast microscope. In some cases, the specimen was previewed in both microscopes before incineration. The results do not support a previous report that the mineral elements of the spore are confined to a peripheral layer, but rather indicate that the spore core as well as the coat are mineral-rich. The cortex may be deficient in minerals, but the possibility of artifact prevents a clear decision on this point. Incinerated B. megaterium spores show a highly ordered fine structure displaying 100 A periodicity in the ash of the middle layer of the coat. The nature of this structure is discussed, as is the technique which demonstrated it. The fine definition of the ash patterns, particularly those obtained with the low-temperature, excited-oxygen technique, suggests that microincineration may be generally useful in the study of fine structure.

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