scholarly journals Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

1986 ◽  
Vol 34 (11) ◽  
pp. 1477-1485 ◽  
Author(s):  
K E Loesser ◽  
K J Doane ◽  
F J Wilson ◽  
F J Roisen ◽  
S Malamed
Keyword(s):  
Immunoelectron Microscopy ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Protein A ◽  
Neuronal Cell ◽  
Cytoskeletal Proteins ◽  
Modified Technique ◽  
Adrenocortical Cells ◽  
Thin Sections ◽  
Lowicryl K4m

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

scholarly journals Effect of resin use in the post-embedding procedure on immunoelectron microscopy of membranous antigens, with special reference to sensitivity.

1990 ◽  
Vol 38 (11) ◽  
pp. 1687-1691 ◽  
Author(s):  
H Shida ◽  
R Ohga
Keyword(s):  
Special Reference ◽  
Immunoelectron Microscopy ◽  
Epoxy Resins ◽  
Protein A ◽  
Epithelial Tissue ◽  
Thin Sections ◽  
Glycoprotein I ◽  
Methacrylate Monomer ◽  
Lowicryl K4m ◽  
Methyl Methacrylate Monomer

To investigate quantitatively the effect of resins on the sensitivity of immunoelectron microscopy of membranous antigen, ultra-thin sections of bovine epithelial tissue embedded in five different kinds of resins [JB-4 (JB4), LR Gold (LRG), Lowicryl K4M (K4M), Quetol 812 (Q812), and Spurr's (Spurr) resin] were labeled specifically with anti-desmosomal glycoprotein I(DGI) antibody followed by protein A-gold (PAG) conjugates. When we compared the labeling intensity expressed as the number of PAG particles per 500-nm length of the desmosomal region along the membrane, three hydrophilic resins (JB4, LRG, and K4M) showed much greater levels of labeling intensity than did epoxy resins (Q812 and Spurr), which had a negative value. The three hydrophilic resins showed only minor differences in their levels of labeling intensity. The intensity obtained with JB4, which was the highest of the three, was further increased by pretreatment of the ultra-thin sections with methyl methacrylate monomer (MM) for 5 min. On the basis of these results, wide applicability of this new technique for membranous antigens, which have been difficult to detect positively by any previously employed techniques, is suggested.

Non-neuronal cell cultures from dorsal root ganglia of the adult cat: production of schwann-like cell lines

1981 ◽  
Vol 229 (1) ◽  
pp. 163-181 ◽  
Author(s):  
Jean R. Wrathall ◽  
Donald D. Rigamonti ◽  
Mark R. Braford ◽  
Carl C. Kao
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Neuronal Cell ◽  
Neuronal Cell Cultures ◽  
Adult Cat

scholarly journals Ultrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections.

1984 ◽  
Vol 98 (1) ◽  
pp. 358-363 ◽  
Author(s):  
S Fakan ◽  
G Leser ◽  
T E Martin
Keyword(s):  
Protein A ◽  
Nuclear Architecture ◽  
Gold Complex ◽  
Thin Sections ◽  
Border Regions ◽  
Core Proteins ◽  
Interchromatin Granules ◽  
Lowicryl K4m ◽  
Perichromatin Fibrils

The ultrastructural distribution of nuclear ribonucleoproteins (RNP) has been investigated by incubation of thin sections of mouse or rat liver, embedded in Lowicryl K4M or prepared by cryoultramicrotomy, with antibodies specific for RNP. The antibodies were localized by means of a protein A-colloidal gold complex. Anti-small nuclear (sn)RNP antibodies, specific for determinants of the nucleoplasmic snRNP species containing U1, U2, U4, U5, and U6 RNAs, were found associated preferentially with perichromatin fibrils, interchromatin granules, and coiled bodies. This indicates an early association of snRNP with structural constituents containing newly synthesized heterogeneous nuclear RNA. It also suggests a possible structural role of some snRNPs in nuclear architecture. Antibodies against the core proteins of heterogeneous nuclear RNP particles associate preferentially with the border regions of condensed chromatin, and in particular with perichromatin fibrils and some perichromatin granules. These results are discussed in view of recent knowledge about the possible role of nucleoplasmic RNP-containing components in the functions of the cell nucleus.

scholarly journals Effect of particle size on labeling density for catalase in protein A-gold immunocytochemistry.

1988 ◽  
Vol 36 (1) ◽  
pp. 107-109 ◽  
Author(s):  
S Yokota
Keyword(s):  
Particle Size ◽  
Quantitative Analysis ◽  
Rat Liver ◽  
Protein A ◽  
High Sensitivity ◽  
Thin Sections ◽  
Gold Particles ◽  
Effect Of Particle Size ◽  
Lowicryl K4m ◽  
Liver Peroxisomes

Effect of particle size on labeling intensity in protein A-gold immunocytochemistry was studied. Catalase labeling of rat liver peroxisomes was used as a labeling model. Ultra-thin sections of Lowicryl K4M-embedded rat liver were stained for catalase with protein A-gold (pAg) probes. Five different sizes of colloidal gold probes, from 5 nm to 38 nm in diameter, were prepared. Labeling intensity decreased as the particle size of the pAg probes increased. The highest labeling was obtained by the 5-nm pAg probe and the lowest by the 38-nm pAg probe. Quantitative analysis also showed that labeling density was inversely proportional to the size of gold particles. The results suggest that the pAg probe with small gold particles has high sensitivity.

scholarly journals Immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells.

1985 ◽  
Vol 100 (1) ◽  
pp. 118-125 ◽  
Author(s):  
J Roth ◽  
M J Lentze ◽  
E G Berger
Keyword(s):  
Plasma Membrane ◽  
Protein A ◽  
Thin Sections ◽  
Golgi Membranes ◽  
Basal Plasma ◽  
Basolateral Plasma Membrane ◽  
Junctional Complexes ◽  
Lowicryl K4m ◽  
Monospecific Antibodies ◽  
Absorptive Enterocytes

Galactosyltransferase immunoreactive sites were localized in human duodenal enterocytes by the protein A-gold technique on thin sections from low temperature Lowicryl K4M embedded biopsy specimens. Antigenic sites detected with affinity-purified, monospecific antibodies were found at the plasma membrane of absorptive enterocytes with the most intense labeling appearing along the brush border membrane. The lateral plasma membrane exhibited a lower degree of labeling at the level of the junctional complexes but the membrane interdigitations were intensely labeled. The labeling intensity decreased progressively towards the basal part of the enterocytes and reached the lowest degree along the basal plasma membrane. Quantitative evaluation of the distribution of gold-particle label proved its preferential orientation to the outer surface of the plasma membrane. In addition to this membrane-associated labeling, the glycocalyx extending from the microvillus tips was heavily labeled. Occasionally, cells without plasma membrane labeling were found adjacent to positive cells. The demonstration of ecto-galactosyltransferase on membranes other than Golgi membranes precludes its general use as a marker for Golgi membrane fractions. The possible function of galactosyltransferase on a luminal plasma membrane is unclear at present, but a role in adhesion appears possible on the basolateral plasma membrane.

scholarly journals THE FINE STRUCTURE OF NEURONS

1955 ◽  
Vol 1 (1) ◽  
pp. 69-88 ◽  
Author(s):  
Sanford L. Palay ◽  
George E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Fine Structure ◽  
Electron Microscope ◽  
Cerebellar Cortex ◽  
Dorsal Root Ganglia ◽  
Medulla Oblongata ◽  
Dorsal Root ◽  
Thin Sections ◽  
Lipid Inclusions ◽  
Nissl Substance

1. Thin sections of representative neurons from intramural, sympathetic and dorsal root ganglia, medulla oblongata, and cerebellar cortex were studied with the aid of the electron microscope. 2. The Nissl substance of these neurons consists of masses of endoplasmic reticulum showing various degrees of orientation; upon and between the cisternae, tubules, and vesicles of the reticulum lie clusters of punctate granules, 10 to 30 mµ in diameter. 3. A second system of membranes can be distinguished from the endoplasmic reticulum of the Nissl bodies by shallower and more tightly packed cisternae and by absence of granules. Intermediate forms between the two membranous systems have been found. 4. The cytoplasm between Nissl bodies contains numerous mitochondria, rounded lipid inclusions, and fine filaments.

Granular cells: A new indicator of neuronal cell death in the spinal ganglia of the rat

1994 ◽  
Vol 52 ◽  
pp. 34-35
Author(s):  
Kevin M. Imel ◽  
J. Franklin Bailey ◽  
Mark DeSantis
Keyword(s):  
Dorsal Root Ganglia ◽  
Axonal Regeneration ◽  
Dorsal Root ◽  
Osmium Tetroxide ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Spinal Ganglia ◽  
Indirect Methods ◽  
Rat Pups ◽  
Dense Bodies

Damage to axons in the neonatal mammal results in the death of the affected neurons rather than survival and axonal regeneration as generally seen in the adult. This phenomenon has been well documented using indirect methods such as counting cells in histological sections of the experimentally damaged versus control tissue. Recent experiments have shown that cells with osmiophilic dense bodies, which have been termed “granules”, become apparent at the light microscopic level in ipsilateral lumbar dorsal root ganglia after destruction of the sciatic nerve on that side in neonatal rats. Within 84 - 96 hours after the lesion, the rat pups were anesthetized, perfused with 4% paraformaldehyde and the ganglia post-fixed in 1% unbuffered osmium tetroxide for at least 72 hours. Such a lengthy exposure to osmium was found to be necessary to visualize the granules.This offers the possibility of using these granules as a direct indicator of neuronal death in the lumbar dorsal root ganglia. The cells containing these granules were usually chromatolytic neuronal somata which had an eccentrically positioned nucleus (when a nucleus or nuclear remnant was still present) and peripheral aggregation of the rough endoplasmic reticulum (i.e. Nissl bodies).

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