Immunoelectron Microscopic Study of Glomerular Lesions Using a Postembedding Method with a Protein A-Gold Complex

Nephron ◽  
1987 ◽  
Vol 46 (2) ◽  
pp. 182-187 ◽  
Author(s):  
Mitsuru Nakajima ◽  
Tadaomi Hirota ◽  
Kiyoaki Kusumoto ◽  
Kouji Taira ◽  
Hidekazu Kamitsuji
Keyword(s):  
Microscopic Study ◽  
Protein A ◽  
Gold Complex ◽  
Glomerular Lesions

The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

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 Electron microscopy of gold-labeled human and equine chromosomes.

1989 ◽  
Vol 37 (9) ◽  
pp. 1443-1447 ◽  
Author(s):  
P E Messier ◽  
R Drouin ◽  
C L Richer
Keyword(s):  
Electron Microscopy ◽  
Chromosome Banding ◽  
Protein A ◽  
Gold Complex ◽  
Chromosome Identification ◽  
Chromosomal Dna ◽  
Metaphase Chromosomes ◽  
Sister Chromatids ◽  
Antigen Localization ◽  
Chromatin Reorganization

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.

scholarly journals Localization of lysosomal and peroxisomal enzymes in the specific granules of rat intestinal eosinophil leukocytes revealed by immunoelectron microscopic techniques.

1984 ◽  
Vol 32 (3) ◽  
pp. 267-274 ◽  
Author(s):  
S Yokota ◽  
H Tsuji ◽  
K Kato
Keyword(s):  
Quantitative Analysis ◽  
Intestinal Mucosa ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Protein A ◽  
Gold Complex ◽  
Double Labeling ◽  
Specific Granules ◽  
Acyl Coa Oxidase ◽  
Microscopic Techniques

Immunoelectron microscopic localization of lysosomal and peroxisomal enzymes in the eosinophil leukocytes of rat intestinal mucosa was studied by use of rabbit antibodies to the enzymes coupled to protein A-gold complex. Gold particle labeling for the lysosomal enzymes, beta-glucuronidase and cathepsin D, was present on specific granules, with a heavy concentration on their paracrystalline cores. The peroxisomal enzymes, acyl-CoA oxidase and catalase, were also found on these granules. The double labeling procedures using two different combination of anti-acyl-CoA oxidase and anti-beta-glucuronidase or anti-catalase and anti-cathepsin D revealed that these enzymes were simultaneously present in specific granules of the intestinal eosinophils. Quantitative analysis of the labeling on subcellular compartments confirmed that all enzymes examined are significantly localized within specific granules and that there is no significant labeling on other compartments such as the nucleus and cytoplasm. In the control sections incubated with an immunoglobulin G fraction from nonimmunized rabbits, no specific labeling was seen on the granules or other organelles. These findings indicate that enzymes which previously have been identified in some organs as lysosomal and in others as peroxisomal can be found together in eosinophil granules.

scholarly journals Use of the protein A-gold technique for the morphological study of vascular permeability.

1980 ◽  
Vol 28 (11) ◽  
pp. 1251-1254 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Endothelial Cells ◽  
Vascular Permeability ◽  
Ultrastructural Study ◽  
Morphological Study ◽  
Protein A ◽  
Gold Complex ◽  
Buffer Solution ◽  
General Applicability ◽  
Immunocytochemical Technique ◽  
Tissue Sections

The protein A-gold immunocytochemical technique has been applied for the ultrastructural study of vascular permeability. A capillary preparation was perfused for 30 min with a buffer solution containing albumin. After fixation and embedding, the albumin molecules were revealed on the tissue sections using anti-albumin antiserum and the protein A-gold complex. A specific labeling was obtained over the capillary lumen, the vesicular structures of the endothelial cells, and the intercapillary space. These results allowed us to conclude that the protein A-gold technique is suitable and of general applicability for the morphological study of vascular permeability.

scholarly journals Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique.

1985 ◽  
Vol 33 (12) ◽  
pp. 1247-1251 ◽  
Author(s):  
C Manigley ◽  
J Roth
Keyword(s):  
Light Microscopy ◽  
Silver Staining ◽  
Colloidal Gold ◽  
Protein A ◽  
Staining Technique ◽  
Gold Complex ◽  
Double Staining ◽  
Red Color ◽  
Gold Immunolocalization ◽  
Red Coloration

We report the development of a new light-microscopic double-staining technique using colloidal gold as sole marker. The contrasting color to the red of colloidal gold is achieved by the application of photochemical silver reaction. The silver reaction, which is principally performed at the end of the first staining sequence, converts the red color of a gold-labeled reagent into black. This contrasts clearly with the red coloration that results from the second incubation sequence without silver reaction. For antigen double staining, the same protein A-gold complex can be used to provide the black and the red color, thus rendering the technique very economical. Alternatively, combination of protein A-gold immunolocalization and lectin-gold staining is possible, as is combined lectin-gold staining.

scholarly journals Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells.

1995 ◽  
Vol 43 (1) ◽  
pp. 11-19 ◽  
Author(s):  
D Malide ◽  
N G Seidah ◽  
M Chrétien ◽  
M Bendayan
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Protein A ◽  
Gold Complex ◽  
Immunocytochemical Study ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Endoproteolytic Cleavage

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin.

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