scholarly journals Ultrastructural localization of intracellular immunoglobulins in Epon-embedded human lymph nodes. An immunoelectron microscopic investigation using the immunogold staining (IGS) and the avidin-biotin-peroxidase complex (ABC) methods.

1985 ◽  
Vol 33 (5) ◽  
pp. 400-406 ◽  
Author(s):  
G Viale ◽  
P Dell'Orto ◽  
P Braidotti ◽  
G Coggi
Keyword(s):  
Lymph Nodes ◽  
Proteolytic Enzymes ◽  
Ultrastructural Localization ◽  
Microscopic Investigation ◽  
Staining Procedure ◽  
Cell Organelles ◽  
Immunogold Staining ◽  
Highly Sensitive ◽  
Immunocytochemical Techniques ◽  
Ultrathin Sections

The ultrastructural localization of intracellular immunoglobulins on ultrathin sections of glutaraldehyde-fixed, postosmicated, and Epon-embedded human lymph nodes has been achieved using such highly sensitive immunocytochemical techniques as immunogold staining and avidin-biotin-peroxidase complex. These immunoelectron microscopic techniques allow the identification of intracellular immunoglobulins without affecting the ultrastructural morphology of the tissue, since they do not require any pretreatment of the sections with proteolytic enzymes or deresinating agents. Therefore, immunoglobulins can be precisely localized in the cell organelles; structures whose morphology is well preserved. The availability of a reliable postembedding staining procedure for the ultrastructural localization of immunoglobulins is of definite value for investigations on human lymphoid tissue, both normal and pathological.

Ultrastructural Changes in the Root Meristem Cells of Rubus Chamaemorus L. (Cloudberry)

1975 ◽  
Vol 33 ◽  
pp. 562-563
Author(s):  
Arya K. Bal
Keyword(s):  
Root Meristem ◽  
Uranyl Acetate ◽  
Particulate Material ◽  
Ultrastructural Changes ◽  
Cell Organelles ◽  
Cytoplasmic Matrix ◽  
Golgi Membranes ◽  
Rubus Chamaemorus ◽  
Dense Substance ◽  
Ultrathin Sections

In the course of studies in the root meristem tissue of Rubus chamaemorus L. some important changes in the ultrastructural morphology were observed during the initiation of senescence at the end of the growing season.Root meristems were collected from naturally growing healthy populations of Cloudberry plants, and fixed in Karnovsky's mixture or in 2.5% glutaraldehyde in phosphate buffer. The samples were osmicated, dehydrated following usual methods and embedded in Epon. Ultrathin sections were stained in uranyl acetate and lead citrate.Figure 1 shows part of a dense cell in the meristem. The electron density of these cells is due to large amounts of a particulate material in the cytoplasmic matrix. The smallest particle seen in electron micrographs is about 40 A, although larger aggregates are also found, which remain randomly distributed in association with various cell organelles. Dense substance has been found associated with golgi membranes, proplastids, vacuoles and microtubules (Fig. 2).

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

scholarly journals Macrophage Autophagy and Oxidative Stress: An Ultrastructural and Immunoelectron Microscopical Study

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Ida Perrotta ◽  
Valentina Carito ◽  
Emilio Russo ◽  
Sandro Tripepi ◽  
Saveria Aquila ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Defense Mechanisms ◽  
Ultrastructural Localization ◽  
Microscopical Study ◽  
Beclin 1 ◽  
Cell Organelles ◽  
A Cell ◽  
First Time

The word autophagy broadly refers to the cellular catabolic processes that lead to the removal of damaged cytosolic proteins or cell organelles through lysosomes. Although autophagy is often observed during programmed cell death, it may also serve as a cell survival mechanism. Accumulation of reactive oxygen species within tissues and cells induces various defense mechanisms or programmed cell death. It has been shown that, besides inducing apoptosis, oxidative stress can also induce autophagy. To date, however, the regulation of autophagy in response to oxidative stress remains largely elusive and poorly understood. Therefore, the present study was designed to examine the ratio between oxidative stress and autophagy in macrophages after oxidant exposure (AAPH) and to investigate the ultrastructural localization of beclin-1, a protein essential for autophagy, under basal and stressful conditions. Our data provide evidence that oxidative stress induces autophagy in macrophages. We demonstrate, for the first time by immunoelectron microscopy, the subcellular localization of beclin-1 in autophagic cells.

Ultrastructural localization of nitric oxide synthase in canine small intestine and colon

1994 ◽  
Vol 266 (4) ◽  
pp. C981-C989 ◽  
Author(s):  
I. Berezin ◽  
S. H. Snyder ◽  
D. S. Bredt ◽  
E. E. Daniel
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Substance P ◽  
Circular Muscle ◽  
Ultrastructural Localization ◽  
Muscle Layer ◽  
Immunogold Staining ◽  
Nitric Oxide Release ◽  
Granular Vesicles ◽  
Oxide Synthase

The ultrastructural distribution and subcellular localization of nitric oxide synthase (NOS) immunoreactivity and its possible colocalization with vasoactive intestinal polypeptide (VIP) and substance P in the muscularis externa in canine ileum and colon were studied by using polyclonal antisera raised against VIP, substance P, and cerebellar NOS. Immunogold staining, with or without silver enhancement, was carried out directly on ultrathin sections using single and two-faced double immunogold methods. NOS immunoreactivity was observed in nerve profiles in myenteric plexus and circular muscle layer. Immunoreactivity was occasionally detected in smooth muscle cells and interstitial cells of Cajal. The double immunostaining revealed NOS and VIP in the same nerve varicosities but never in the same organelles. NOS was localized in electron-dense material of undetermined nature, whereas VIP was associated with large granular vesicles. Substance P and NOS were never found in the same nerves. These results indicate that NOS is present in the enteric nerves containing VIP but in different organelles and that nitric oxide release probably does not occur by an exocytotic mechanism.

scholarly journals Ultrastructural localization of nuclear matrix proteins in HeLa cells using silver-enhanced ultra-small gold probes.

1991 ◽  
Vol 39 (8) ◽  
pp. 1035-1045 ◽  
Author(s):  
A de Graaf ◽  
P M van Bergen en Henegouwen ◽  
A M Meijne ◽  
R van Driel ◽  
A J Verkleij
Keyword(s):  
Hela Cells ◽  
Nuclear Matrix ◽  
Matrix Protein ◽  
Ultrastructural Localization ◽  
Matrix Proteins ◽  
Cell Permeabilization ◽  
Immunogold Staining ◽  
Thin Sections ◽  
Nuclear Matrix Proteins ◽  
Dna And Rna

We describe a method for immunogold staining of nuclear matrix proteins using ultra-small gold particles. The nuclear matrix of HeLa cells is obtained by two fractionation steps: (a) cell permeabilization with Triton X-100 to isolate the cytoskeleton, and (b) nuclease digestion followed by an incubation in 0.25 M ammonium sulfate to isolate the nuclear matrix. To prevent redistribution of internal matrix proteins during nuclear matrix preparation, pre-fixation with 0.1% acrolein was performed. Under this condition up to 80% of protein and 90% of DNA and RNA could be removed on nuclear matrix isolation, without redistribution of internal nuclear matrix proteins. For immunogold labeling, 1-nm gold probes appeared to be required to obtain optimal penetration into the nucleus. These particles can be visualized after silver enhancement. After gold labeling the matrices are stained, embedded in Epon, and ultra-thin sections are prepared for examination in the electron microscope. The applicability of this method is examplified by the localization of a 125 KD internal nuclear matrix protein and the lamins A and C in nuclear matrix preparations of HeLa cells.

scholarly journals THE ULTRASTRUCTURAL LOCALIZATION OF CYTOCHROME c-OXIDASE COMPLEX IN A LIPID-RETAINING, GLUTARALDEHYDE-UREA EMBEDMENT

1974 ◽  
Vol 22 (11) ◽  
pp. 1019-1027 ◽  
Author(s):  
IZHAK NIR ◽  
DANIEL C. PEASE
Keyword(s):  
Ethylene Glycol ◽  
Kidney Tissue ◽  
Ultrastructural Localization ◽  
Precise Localization ◽  
Reaction Products ◽  
Mitochondrial Membranes ◽  
En Bloc ◽  
Fresh Tissue ◽  
Fixed Tissue ◽  
Ultrathin Sections

Kidney tissue, incubated in a phosphate-sucrose buffer with diaminobenzidine (DAB), subsequently was embedded in polymerized glutaraldehyde-urea (Pease and Peterson, 1972). The highly polar character of this embedment retains lipids in ultrathin sections and thus permits a precise localization of reaction products in relation to cytomembranes. Furthermore, since conventional organic solvents are not used during processing, it is thought that oxidized DAB polymers certainly remain in place. Their density can be enhanced by exposing mounted sections to OsO4 vapor, rather than by en bloc staining. DAB oxidation takes place only in the compartment between the inner and outer mitochondrial membranes. When aldehyde-fixed tissue is incubated, the deposits are largely limited to the intracristal spaces, whereas when fresh tissue is incubated, the entire compartment is uniformly filled. Morphologic features of fresh, unfixed tissue are stabilized by ethylene glycol and so survive incubation best when about 30% of this substance is added to the medium.

scholarly journals Immunoelectron microscopic demonstration of S-100b protein-like in centriole, cilia, and basal body.

1988 ◽  
Vol 36 (6) ◽  
pp. 693-696 ◽  
Author(s):  
T Uchida ◽  
T Endo
Keyword(s):  
Basal Body ◽  
Colloidal Gold ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Basal Bodies ◽  
Cell Organelles ◽  
Gold Particles ◽  
Microscopic Demonstration ◽  
S 100 ◽  
And Function

We report here the ultrastructural localization of S-100b protein-like immunoreactivity in the centriole, cilia, and basal body. Duodenum and trachea of guinea pigs and rats were fixed and immunostained by the protein A-gold method. All centrioles, cilia, and basal bodies observed showed clear S-100b protein-like immunoreactivity. Specific colloidal gold particles were located over the microtubules in these cell organelles. However, other microtubules scattered throughout the cytoplasm were devoid of immunoreactivity. Although the functional significance of S-100b protein-like immunoreactivity in the centriole, cilia, and basal bodies remains to be elucidated, the present results introduce new perspectives into the investigation of localization and function of S-100 proteins.

scholarly journals Deposition of kappa and lambda light chains in amyloid filaments of dialysis-related amyloidosis.

1995 ◽  
Vol 6 (4) ◽  
pp. 1262-1270
Author(s):  
D Brancaccio ◽  
G M Ghiggeri ◽  
P Braidotti ◽  
A Garberi ◽  
M Gallieni ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Water Extraction ◽  
Major Constituent ◽  
Light Chains ◽  
Staining Procedure ◽  
Immunogold Staining ◽  
Amino Terminal ◽  
Amyloid Deposits

beta 2-Microglobulin (beta 2m) is considered to be the amyloidogenic precursor in dialysis-related amyloidosis, although the implication of other relevant cofactors in the pathogenesis of this disease has also been hypothesized. It is conceivable that substances found in amyloid deposits might represent something more than simple codeposition, possibly playing a pathogenic role in amyloidogenesis. Along these lines, a detailed analysis of the protein composition of amyloid fibrils purified from synovial material surgically obtained from nine patients on long-term dialysis was carried out. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several other protein components, in addition to beta 2m, were found. These were characterized by NH2 amino-terminal sequencing and immunoblotting. In fibrils obtained by water extraction, which fulfill the electron microscopy criteria of highly pure amyloid material, polyclonal kappa and lambda light chains were detected with a concentration of 15 micrograms/mL in the water extraction material; the beta 2m concentration was 200 micrograms/mL. Light microscopy immunohistochemistry was performed on samples from five patients. Amyloid deposits reacted with anti-beta 2m, and anti-light (kappa, lambda), chain antibodies. The immunoreaction of amyloid filaments to anti-beta 2m, anti-lambda, and anti-kappa light chain antibodies was also tested by electron microscopy by use of the immunogold staining procedure. Amyloid filaments were labeled by the three antibodies and showed a different intensity of immunostaining apparently related to their different aggregation pattern. These observations demonstrate that polyclonal immunoglobulin light chains (kappa and lambda) are not contaminants but, together with beta 2m, represent a major constituent of amyloid deposits in dialysis-related osteoarticular amyloidosis, thus indicating their possible role in amyloidogenesis.

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