scholarly journals Ultrastructural localization of basic proteins in Trypanosoma cruzi.

1978 ◽  
Vol 26 (5) ◽  
pp. 349-358 ◽  
Author(s):  
T Souto-Padrón ◽  
W De Souza
Keyword(s):  
Trypanosoma Cruzi ◽  
Phosphotungstic Acid ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Basic Proteins ◽  
Intense Reaction ◽  
Cytoplasmic Vacuoles ◽  
Ammoniacal Silver

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) techniques were applied in epimastigote and trypomastigote forms of the pathogenic protozoa Trypanosoma cruzi to detect basic proteins at the ultrastructural level. With both techniques, reaction was observed in the nucleus and in some cytoplasmic vacuoles. In the kinetoplast of epimastigotes, reaction was observed only at its periphery. In trypomastigotes, however, an intense reaction was observed in the spherical kinetoplast. With the ethanolic phosphotungstic acid technique, reaction was also observed in ribosomes and at the peripheral doublet microtubules of the flagellum. The filaments which form the paraflagellar structure did not react.

scholarly journals Ultrastructural localization of basic proteins in cytoplasmic granules of rat eosinophils and mast cells.

1980 ◽  
Vol 28 (3) ◽  
pp. 238-244 ◽  
Author(s):  
P F Pimenta ◽  
M A Loures ◽  
W de Souza
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Basic Protein ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Cytoplasmic Granules ◽  
Basic Proteins ◽  
Intense Reaction ◽  
The Matrix ◽  
Ammoniacal Silver

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) methods were used to localize basic protein at the ultrastructural level in cytoplasmic granules of rat eosinophils and mast cells isolated using a Metrizamide gradient. Intense reaction was seen in the granules of EPTA-treated eosinophils. Following incubation of the cells for 2 hr in EPTA alone, the matrix was stained. After longer incubation (10 hr), however, both the matrix and core were stained. Cytoplasmic granules of the mast cell show a slight or negative reaction with EPTA. With the AS technique, a large number of silver particles were seen in the nucleus of both eosinophils and mast cells. The mast cell cytoplasmic granules showed intense reaction, while those from eosinophils showed no clear reaction. Acetylation of the cells under conditions sufficient to block most free amino groups prio to EPTA or AS treatment greatly reduced (EPTA) or abolished (AS) the reaction. The results indicate 1) that eosinophil granules contain basic proteins both in the matrix and the core, 2) that the mast cell granules contain a basic protein (probably the alpha-chymotrypsin-like enzyme), which reacts strongly with AS, and 3) that the AS and EPTA methods have different specificities.

Ultrastructural localization of cationic proteins in human polymorphonuclear leukocytes

1978 ◽  
Vol 30 (1) ◽  
pp. 21-35
Author(s):  
W.J. Brown ◽  
E.M. Wood
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Osmium Tetroxide ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Cationic Proteins ◽  
Cytoplasmic Granules ◽  
Mediators Of Inflammation ◽  
Human Polymorphonuclear Leukocytes ◽  
Ammoniacal Silver ◽  
Neutral Proteases

The present investigation is concerned with the use of the post-formalin ammoniacal silver reaction to detect the arginine-rich cationic proteins in human polymorphonuclear leukocytes at the ultrastructural level. These proteins appear to function as neutral proteases in antibacterial action and as mediators of inflammation. Originally, the ammoniacal silver reaction relied upon primary fixation in dilute formalin which prevented optimum fixation of tissues. This study shows that by using the proper sequence of glutaraldehyde fixation and the ammoniacal silver solution in conjunction with osmium tetroxide treatment, better fixation of the tissue and localization of the ammoniacal silver reaction can be achieved. Also, the ammoniacal silver reaction in human polymorphonuclear leukocytes is exclusively located in the large, crystalline cytoplasmic granules of eosiniphils. All other cytoplasmic granules of neutrophils, eosinophils, and basophils were found to be devoid of the ammoniacal silver reaction product. These results are contrary to previously published experimental data. Possible explanations for this discrepancy are discussed.

Ultrastructural Localization of Antigens by Capillary Action Immunocytochemistry

1996 ◽  
Vol 54 ◽  
pp. 40-41
Author(s):  
László G. Kömüves
Keyword(s):  
San Francisco ◽  
Colloidal Gold ◽  
Specific Binding ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Adhesive Tape ◽  
Distilled Water ◽  
Capillary Action ◽  
Rabbit Igg

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.

Ultrastructural localization of cationic proteins in cytoplasmic granules of chicken and rabbit polymorphonuclear leukocytes

1975 ◽  
Vol 17 (1) ◽  
pp. 79-94
Author(s):  
E.K. Macrae ◽  
J.K. Spitznagel
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Intracellular Localization ◽  
Ultrastructural Localization ◽  
Cationic Protein ◽  
Cationic Proteins ◽  
Cytoplasmic Granules ◽  
Electron Dense Deposit ◽  
Dense Deposit ◽  
Neutrophilic Polymorphonuclear Leukocytes ◽  
Ammoniacal Silver

Cytoplasmic granules known to contain cationic arginine-rich proteins can be identified by the ammoniacal silver reaction (ASR) which provides a cytochemical marker detectable under the electron microscope. Only the large rod-shaped granules of the chicken polymorphonuclear leukocytes (heterophils) and the large spherical azurophilic granules of the rabbit neutrophilic polymorphonuclear leukocytes show the ASR product as a discrete particulate electron-dense deposit. The other smaller granules are devoid of reaction product, as are membranes and mitochondria. The intracellular localization of the ASR product, as are membranes and mitochondria. The intracellular localization of the ASR product on the large granules coincides with the ASR product localization on the same isolated granule populations, when the ammoniacal silver reaction is applied to these granules after their separation by sucrose-density gradients. The cationic proteins may have intraleukocytic bacteriolytic properties, since ASR product, presumably indicating cationic protein from discharged granules, appears to surround ingested bacteria within cytoplasmic phagosomes.

scholarly journals The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin.

1986 ◽  
Vol 34 (8) ◽  
pp. 1069-1077 ◽  
Author(s):  
K Hedman ◽  
I Pastan ◽  
M C Willingham
Keyword(s):  
Plasma Membrane ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Coated Pits ◽  
Golgi Stack ◽  
Acetylneuraminic Acid ◽  
Secretory Glycoproteins ◽  
Double Label ◽  
Endocytic Vesicles ◽  
Exocytic Pathway

The subcellular distribution of sialic acid was determined at the ultrastructural level using Limax flavus agglutinin (LFA). This lectin, which is specific for N-acetylneuraminic acid and N-glycolylneuraminic acid, was covalently conjugated to horseradish peroxidase (HRP). The conjugates (LFA-HRP) were applied to aldehyde-fixed, saponin-permeabilized 3T3 cells in pre-embedding labeling electron microscopy. Peroxidase label was detected in a patchy distribution at the cell surface, and in plasma-membrane-coated pits, endocytic vesicles (receptosomes), multivesicular bodies, and lysosomes. Smooth-surfaced tubular and vesicular structures, similar to those that participate in membrane recycling, were labeled. In the Golgi complex, more than half of the cisternae contained label--typically only one cisterna on the cis side was unlabeled. Heavily labeled structures of the trans Golgi included a reticular membranous system with coated regions--50-80 nm diameter vesicular or pit-like profiles and larger coated vacuoles. Smooth 200-300 nm vacuoles were labeled on the trans side of the Golgi stack. Similar structures have been previously shown to participate in the exocytosis of plasma membrane and secretory glycoproteins from the Golgi stacks. These findings identify those intracellular organelles that are functionally at the level of, or distal to, the sialyltransferase-containing membranes of the Golgi, and distinguish them from the pre-Golgi membranous structures. The LFA-HRP conjugate is an indicator for this functional trans domain of the cell, and should be applicable for ultrastructural double-label experiments as a cis versus trans marker of the exocytic pathway.

Tritrichomonas foetus: Ultrastructural localization of basic proteins and carbohydrates

1982 ◽  
Vol 54 (2) ◽  
pp. 135-144 ◽  
Author(s):  
Marlene Benchimol ◽  
Cezar A. Elias ◽  
Wanderley de Souza
Keyword(s):  
Ultrastructural Localization ◽  
Tritrichomonas Foetus ◽  
Basic Proteins

Ultrastructural localization of dystropin in human muscle by using gold immunolabelling

1990 ◽  
Vol 240 (1297) ◽  
pp. 197-210 ◽  
Keyword(s):  
Plasma Membrane ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Human Muscle ◽  
Muscle Fibres ◽  
Mouse Muscle ◽  
Transverse Tubular System ◽  
Cytoplasmic Face ◽  
Tubular System ◽  
Gold Probe

Immunolabelling with a 5 nm gold probe was used to localize dystrophin at the ultrastructural level in human muscle. The primary antibody was monoclonal, raised against a segment (amino acids 1181-1388) from the rod domain of dystrophin. The antibody (Dy4/6D3) is specific for dystrophin and shows no immunoreactivity with any protein from mdx mouse muscle or from patients with a gene deletion spanning part of the molecule recognized by the antibody (Nicholson et al . 1989 a ; England et al . 1990). Using this antibody, labelling was almost entirely confined to a narrow 75 nm rim at the periphery of the muscle fibres. Histograms of the distance from the gold probe to the cytoplasmic face of the plasma membrane and of the distance between gold probes (nearest neighbour in a plane parallel with the plasma membrane) displayed modes at approximately 15 nm and 120 nm, respectively. The distribution of the probe was the same in longitudinal and transverse sections of the muscle. These observations suggest that the rod portion of the dystrophin mole­cule is normally arranged close to the cytoplasmic face of the plasma membrane and that the molecules form an interconnecting network. Labelling was not associated with the transverse tubular system.

scholarly journals A new lectin-gold complex for ultrastructural localization of galacturonic acids.

1988 ◽  
Vol 36 (11) ◽  
pp. 1403-1411 ◽  
Author(s):  
N Benhamou ◽  
N Gilboa-Garber ◽  
J Trudel ◽  
A Asselin
Keyword(s):  
Binding Sites ◽  
Ricinus Communis ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Gold Complex ◽  
Carbohydrate Binding ◽  
Wheat Coleoptile ◽  
Ultrastructural Studies ◽  
Pectic Substances ◽  
Considerable Loss

We report the development of a cytochemical affinity technique for detection of galacturonic acids at the ultrastructural level. The highly purified gonad lectin from Aplysia depilans (AGL) was tagged with colloidal gold particles and used for labeling carbohydrates in resin-embedded sections of various plant and fungal tissues. Patterns of AGL binding sites were compared to those obtained with a D-galactose-specific lectin, Ricinus communis agglutinin I. Differences in labeling patterns were noted, indicating that the lectins exhibited differential carbohydrate binding. In addition, the considerable loss of labeling over isolated wheat coleoptile walls treated for removal of pectin, after incubation with the AGL-gold complex, strongly suggested an affinity of AGL for pectic substances. A series of cytochemical controls, including sugar inhibition tests, has proven the specificity of the technique and the high affinity of AGL towards galacturonic acids. The potential value of this new lectin for ultrastructural studies on cell wall pectic substances in plant biology and pathology is demonstrated.

scholarly journals Heterogeneity of uroplakin localization in human normal urothelium, papilloma and papillary carcinoma

2013 ◽  
Vol 47 (4) ◽  
pp. 338-345 ◽  
Author(s):  
Dasa Zupancic ◽  
Rok Romih
Keyword(s):  
Papillary Carcinoma ◽  
Recurrence Rate ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Apical Plasma Membrane ◽  
High Recurrence Rate ◽  
Tumour Resection ◽  
Urothelial Cells ◽  
Normal Human ◽  
Normal Urothelium

Abstract Background. Uroplakins are differentiation-related membrane proteins of urothelium. We compared uroplakin expression and ultrastructural localization in human normal urothelium, papilloma and papillary carcinoma. Because of high recurrence rate of these tumours, treated by transurethral resection, we investigated urothelial tumour, resection border and uninvolved urothelium. Patients and methods. Urinary bladder samples were obtained from tumour free control subjects and patients with papilloma and papillary carcinoma. Immunohistochemical and immunoelectron labelling of uroplakins were performed. Results. In normal human urothelium with continuous uroplakin-positive superficial cell layer uroplakins were localized to flattened mature fusiform vesicles and apical plasma membrane of umbrella cells. Diverse uroplakin expression was found in papilloma and papillary carcinoma. Three aberrant differentiation stages of urothelial cells, not found in normal urothelium, were recognized in tumours. Diverse uroplakin expression and aberrant differentiation were occasionally found in resection border and in uninvolved urothelium. Conclusions. We demonstrated here that uroplakin expression and localization in urothelial tumours is altered when compared to normal urothelium. In patients with papilloma and papillary carcinoma immunolabelling of uroplakins at ultrastructural level shows aberrant urothelial differentiation. It is possible that aberrant differentiation stages of urothelial cells in resection border and in uninvolved urothelium contribute to high recurrence rate.

scholarly journals Visualization of anti-sperm plasma membrane IgG and Fab as a method for localization of boar sperm membrane antigens.

1982 ◽  
Vol 30 (12) ◽  
pp. 1217-1227 ◽  
Author(s):  
L D Russell ◽  
R N Peterson ◽  
T A Russell
Keyword(s):  
Plasma Membrane ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Ultrastructural Localization ◽  
Surface Antigens ◽  
Ultrastructural Level ◽  
Simple Method ◽  
Sperm Surface ◽  
Sperm Plasma Membrane ◽  
Sperm Antigens

A simple method for ultrastructural localization of sperm surface antigens by direct visualization of bound antibodies is presented. Anti-sperm plasma membrane (ASPM) immunoglobulin (Ig) G, visualized in tissues treated with an osmium:ferrocyanide mixture, projected 11-13 nm from the surface and ASPM Fab fragments projected 8-10 nm from the surface. The density of IgG labeling, as subjectively estimated, corresponded to indirect immune fluorescein isothiocyanate, indirect immunoferritin, and sperm-vesicle labeling patterns. Agglutination of sperm vesicles and sperm were demonstrated and the linking antibody visualized. A second antibody on protein A directed against ASPM IgG made the immunologic tag more apparent and indicated, in disrupted sperm preparations, labeling of both sides of the plasma membrane. The method provides for easy and sensitive localization of sperm surface antigens at the ultrastructural level and is presently being used to localize specific sperm antigens.

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