scholarly journals ELECTRON MICROSCOPIC DEMONSTRATION OF DEHYDROGENASE ACTIVITY WITH A NEW OSMIOPHILIC DITETRAZOLIUM SALT (TC-NBT)

1967 ◽  
Vol 15 (1) ◽  
pp. 1-13 ◽  
Author(s):  
ARNOLD M. SELIGMAN ◽  
HIROMI UENO ◽  
YOSHIHISA MORIZONO ◽  
HANNAH L. WASSERKRUG ◽  
LIONEL KATZOFF ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Dehydrogenase Activity ◽  
Osmium Tetroxide ◽  
Electron Microscopic ◽  
Tissue Sections ◽  
Thin Slices ◽  
Microscopic Demonstration ◽  
Electron Microscopic Demonstration ◽  
Formalin Fixed

Dissatisfaction with the degree of contrast produced by currently available tetrazolium salts in electron microscopy of the dehydrogenases stimulated us to design and prepare an osmiophilic ditetrazolium salt (TC-NBT)3 containing thiocarbamyl groups in innocuous positions of the nitro-BT molecule. The new reagent has the same favorable properties as nitro-BT for light microscopy and, in addition, is convertible to osmium black when tissue sections containing the diformazan are exposed to osmium tetroxide vapor. The stained tissue sections withstand dehydration and embedding in Araldite. Fresh and formalin-fixed rat heart, cut in thin slices without freezing, were stained for SDH and NADH2-D activity. In fresh heart, both dehydrogenases produced dense and thickened cristae in mitochondria that were active. Heterogeneity of mitochondria ris à vis dehydrogenase activity was confirmed. In fresh preparations stained mitochondria were contracted with narrowing of intercristal spaces as compared to inactive mitochondria. This was not the case in formalin-fixed heart. The dehydrogenase activity of individual mitochondria appeared to be an all-or-none phenomenon in these preliminary studies and the diformazan deposits were quite uniformly distributed along both surfaces of the cristae with encroachment on intracristal spaces in heavily stained preparations. The outer limiting membranes of the active mitochondria did not have diformazan deposits in contrast to the heavily stained cristae. Unreactive mitochondria revealed the usual contrast seen in control sections exposed to osmium tetroxide or in deep parts of the blocks to which TC-NBT did not penetrate.

scholarly journals Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus: a cytochemical affinity technique with the use of fetuin-gold complexes.

1984 ◽  
Vol 32 (11) ◽  
pp. 1167-1176 ◽  
Author(s):  
J Roth ◽  
J M Lucocq ◽  
P M Charest
Keyword(s):  
Electron Microscopy ◽  
Sialic Acid ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
Gold Complexes ◽  
Thin Sections ◽  
Tissue Sections ◽  
Lowicryl K4m ◽  
Hydrolysis Of ◽  
Electron Microscopic Demonstration

The development of a cytochemical affinity technique for the demonstration of sialic acid residues by light and electron microscopy is reported. The lectin from the slug Limax flavus, with its narrow specificity for N-acetyl- and N-glycolylneuraminic acid, was applied to tissue sections. Subsequently fetuin-gold complexes were used to visualize the tissue-bound lectin. Different cytochemical controls, including sugar inhibition tests, neuraminidase digestion, the use of fetuin-gold complexes alone, or acid hydrolysis of sections, proved the specificity of the technique. Postembedding staining was performed on frozen, paraffin, or semithin resin sections for light microscopy and on thin sections from low temperature Lowicryl K4M-embedded material for electron microscopy. The distribution of sialic acid residues in rat pancreas, liver, and colonic mucosa was investigated.

scholarly journals Electron microscopic demonstration of neural connections using horseradish peroxidase: a comparison of the tetramethylbenzidine procedure with seven other histochemical methods.

1982 ◽  
Vol 30 (5) ◽  
pp. 425-435 ◽  
Author(s):  
K A Carson ◽  
M M Mesulam
Keyword(s):  
Electron Microscopy ◽  
Spinal Cord ◽  
Motor Neurons ◽  
Lumbar Spinal Cord ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Anterograde Transport ◽  
Microscopic Demonstration ◽  
Lumbar Spinal ◽  
Electron Microscopic Demonstration

Eight methods for the electron microscopic demonstration of horseradish peroxidase (HRP) labeling have been compared in adjacent series of vibratome sections of mouse lumbar spinal cord. The tracer, a HRP-wheat germ agglutinin (WGA) conjugate, was injected into the gastrocnemius muscle complex. Following retrograde axonal transport to the lumbar motor neurons and transganglionic anterograde transport of the tracer to the dorsal horn, the HRP activity was demonstrated in eight series of adjacent sections of lumbar spinal cord using eight methods. These included procedures using tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC), o-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and 4 methods using 3,3'-diaminobenzidine (DAB). All eight methods were able to demonstrate both retrograde labeling of motor neurons and transganglionic anterograde transport into the dorsal horn. However, there were differences in the appearance of the various reaction products under the electron microscope. In addition, differences in the distribution of the reaction products were observed by both light and electron microscopy. The largest distribution of reaction product was observed with TMB. BDHC and o-tolidine were next, followed by the DAB procedures and PPD-PC. The TMB, BDHC, and o-tolidine reaction products were all found to be suitable for electron microscopy. The TMB reaction product was electron dense and had a very distinctive crystalloid appearance that made identification of HRP-labeled neuronal profiles easy and unequivocal.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

A Method for Electron Microscopic Study of Tissue Culture Cell Monolayers Grown in Tubes

1967 ◽  
Vol 25 ◽  
pp. 132-133
Author(s):  
R. Stephens ◽  
G. Schidlovsky ◽  
S. Kuzmic ◽  
P. Gaudreau
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Light Microscopy ◽  
Cell Sheet ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Electron Microscopic ◽  
Cell Sheets ◽  
Morphological Alterations ◽  
Culture Cells

The usual method of scraping or trypsinization to detach tissue culture cell sheets from their glass substrate for further pelletization and processing for electron microscopy introduces objectionable morphological alterations. It is also impossible under these conditions to study a particular area or individual cell which have been preselected by light microscopy in the living state.Several schemes which obviate centrifugation and allow the embedding of nondetached tissue culture cells have been proposed. However, they all preserve only a small part of the cell sheet and make use of inverted gelatin capsules which are in this case difficult to handle.We have evolved and used over a period of several years a technique which allows the embedding of a complete cell sheet growing at the inner surface of a tissue culture roller tube. Observation of the same cell by light microscopy in the living and embedded states followed by electron microscopy is performed conveniently.

scholarly journals FIXATION OF NEURAL TISSUES FOR ELECTRON MICROSCOPY BY PERFUSION WITH SOLUTIONS OF OSMIUM TETROXIDE

1962 ◽  
Vol 12 (2) ◽  
pp. 385-410 ◽  
Author(s):  
Sanford L. Palay ◽  
S. M. McGee-Russell ◽  
Spencer Gordon ◽  
Mary A. Grillo
Keyword(s):  
Electron Microscopy ◽  
Nervous System ◽  
Osmium Tetroxide ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Neural Tissues ◽  
Structural Relations ◽  
Cellular Components

This paper describes in detail a method for obtaining nearly uniform fixation of the nervous system by vascular perfusion with solutions of osmium tetroxide. Criteria are given for evaluating the degree of success achieved in the preservation of all the cellular components of the nervous system. The method permits analysis of the structural relations between cells at the electron microscopic level to an extent that has not been possible heretofore.

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