scholarly journals Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.

1983 ◽  
Vol 31 (1) ◽  
pp. 101-109 ◽  
Author(s):  
M Bendayan ◽  
M Zollinger
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Liver Mitochondria ◽  
Secretory Proteins ◽  
Additional Advantage ◽  
Sodium Metaperiodate ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Structural Preservation

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.

scholarly journals Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.

1978 ◽  
Vol 26 (12) ◽  
pp. 1074-1081 ◽  
Author(s):  
J Roth ◽  
M Bendayan ◽  
L Orci
Keyword(s):  
Endocrine Pancreas ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Gold Complex ◽  
Secretory Proteins ◽  
Staphylococcal Protein A ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Step Procedure ◽  
General Method

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.

Ultrastructural localization of cytoskeletal proteins in pancreatic secretory cells

1985 ◽  
Vol 63 (6) ◽  
pp. 680-690 ◽  
Author(s):  
Moïse Bendayan
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Cytoskeletal Proteins ◽  
Secretory Cells ◽  
Secretory Proteins ◽  
Protein Maturation ◽  
Pancreatic Cells ◽  
Pancreatic Exocrine ◽  
Cell Web

Actin, myosin, and keratin immunoreactive sites have been localized with high resolution in pancreatic exocrine cells, by applying the protein A – gold technique on tissues processed at low temperature conditions. The labeling by gold particles was found at the level of the cell web and closely associated with the limiting membranes of the immature and mature secretory granules, as well as those of the "trans" cisternae of the Golgi apparatus. These results, together with those obtained in the study on the localization of secretory proteins in exocrine pancreatic cells, demonstrate that cytoskeletal proteins are present at sites where maturation and (or) concentration of the secretory proteins occur. Thus, besides the role that cytoskeletal proteins must play in the transport of the secretory granules from the Golgi to the plasma membrane, they may also be involved in the process of protein maturation and (or) concentration.

scholarly journals Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold.

1985 ◽  
Vol 33 (10) ◽  
pp. 1015-1025 ◽  
Author(s):  
M Castel ◽  
J Morris ◽  
Y Ben-Barak ◽  
R Timberg ◽  
N Sivan ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Secretory Granules ◽  
Picric Acid ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Neurosecretory Granules ◽  
Sodium Metaperiodate ◽  
Background Staining ◽  
Axonal Varicosity

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.

Isolation and ultrastructural localization of a soluble protein from Ophiostoma ulmi

1990 ◽  
Vol 68 (11) ◽  
pp. 2517-2524 ◽  
Author(s):  
R. S. Jeng ◽  
A. M. Svircev
Keyword(s):  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Leading Edge ◽  
Ultrastructural Localization ◽  
Immunogold Labelling ◽  
Two Dimensional ◽  
Thin Sections ◽  
Ophiostoma Ulmi ◽  
Gold Label

Two-dimensional polyacrylamide gel electrophoresis was used to identify and isolate a soluble polypeptide, the QP1 protein, which is characteristic of the vegetative hyphae of nonaggressive isolate Q412 of Ophiostoma ulmi. Individual QP1 spots were excised from 16 two-dimensional gels. Polypeptides were eluted from the gel spots by electroelution and lyophilized. The protein was injected into rabbits for the production of polyclonal antibodies. Antiserum specificity was tested by transferring polypeptides from a two-dimensional gel onto nitrocellulose and treating with QP1 serum. The resulting immunoblot contained a single spot that corresponded in shape and location to that of the QP1 polypeptide. Thin sections of fungal mycelia, from nonaggressive isolate Q412 and the aggressive isolate VA of O. ulmi, were treated with QP1 antibodies and protein A – gold. The gold label was localized in thin sections over conidial and hyphal cell walls of the nonaggressive isolate. The aggressive isolate was nonreactive. Mycelia from nonaggressive isolates Q412 and Q311 and aggressive isolates VA and CESS16K of O. ulmi were grown on solid medium, treated with QP1 antibodies, labelled with protein A – gold, and prepared for scanning electron microscopy. The gold-labelled QP1 polypeptide was detected on the leading edge of a small number of hyphae from nonaggressive isolates Q412 and Q311. Key words: immunogold labelling, Ophiostoma ulmi, soluble proteins.

scholarly journals Double immunocytochemical labeling applying the protein A-gold technique.

1982 ◽  
Vol 30 (1) ◽  
pp. 81-85 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Tissue Section ◽  
Protein A ◽  
Antigenic Site ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Double Labeling ◽  
Gold Particles ◽  
Antigenic Sites

In the present study we report the modifications and the different steps of the protein A-gold (pAg) technique that allow the simultaneous demonstration of two antigenic sites on the same tissue section. The labeling is carried out in the following manner: face A of the tissue section is incubated with an antiserum followed by a pAg complex prepared with large gold particles; face B of the same tissue section is then incubated with a second antiserum followed by a pAg complex prepared with small gold particles. Each of the pAg complexes reveals a different antigenic site on opposite faces of the tissue section. The transparency of the section in the electron beam allows the visualization of the gold particles present on both faces. The double labeling pAg technique was applied for the simultaneous demonstration of two secretory proteins in the same Golgi, condensing vacuoles, and zymogen granules of the rat pancreatic acinar cells.

scholarly journals Ultrastructural localization of acidic glycoconjugates with the low iron diamine method.

1982 ◽  
Vol 30 (5) ◽  
pp. 471-476 ◽  
Author(s):  
M Takagi ◽  
R T Parmley ◽  
S S Spicer ◽  
F R Denys ◽  
M E Setser
Keyword(s):  
Bone Marrow ◽  
External Surface ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Electron Microscope Level ◽  
Thin Sections

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.

Endocytosis of Proteins by Salivary Gland Duct Cells

1987 ◽  
Vol 66 (2) ◽  
pp. 412-419 ◽  
Author(s):  
A.R. Hand ◽  
R. Coleman ◽  
M.R. Mazariegos ◽  
J. Lustmann ◽  
L.V. Lotti
Keyword(s):  
Protein A ◽  
Periodate Oxidation ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Cationized Ferritin ◽  
Cell Secretion ◽  
High Concentration ◽  
Thin Sections ◽  
Duct Cells ◽  
Rat Parotid

The ability of the intralobular duct cells of the rat parotid gland to take up protein from the lumen was examined by retrograde infusion of exogenous proteins and by immunogold localization of endogenous secretory proteins. Small amounts of native horseradish peroxidase (HRP) were taken up by intercalated and striated duct cells, and were present in small vesicles, multi vesicular bodies, and lysosomes. In contrast, HRP modified by periodate oxidation was avidly internalized by the duct cells and was present in large apical vacuoles that acquired lysosomal hydrolase activity. Native and cationized ferritin were taken up in a similar manner when infused at a high concentration (up to 10 mg/mL). At lower concentrations (0.3-1.0 mg/mL), endocytosis of cationized ferritin occurred mainly in small apical tubules and vesicles in striated duct cells. Little native ferritin was taken up at these concentrations. After stimulation of acinar cell secretion by isoproterenol, similar vacuoles were occasionally observed in both intercalated and striated duct cells. Labeling of thin sections with antibodies to amylase and to a 26,000-dalton secretory protein (protein B1), followed by protein A-gold, revealed the presence of these proteins in the vacuoles, indicating endocytosis of acinar secretory proteins by the duct cells. Although uptake of acinar proteins by duct cells occurs at a low rate in normal animals, previous work suggests that extensive endocytosis may occur in certain pathological conditions. This may be a mechanism for removing abnormal or modified proteins from saliva before it reaches the oral cavity.

scholarly journals An improved immunocytochemical method for subcellular localization of serotonin in rat enterochromaffin cells.

1987 ◽  
Vol 35 (3) ◽  
pp. 319-326 ◽  
Author(s):  
O Nilsson ◽  
A Dahlström ◽  
M Geffard ◽  
H Ahlman ◽  
L E Ericson
Keyword(s):  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Level ◽  
Proximal Colon ◽  
Intracellular Distribution ◽  
Thin Sections ◽  
Ec Cells ◽  
Immunocytochemical Method ◽  
A Minor

Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.

scholarly journals Electron microscopic localization of chromogranin A in osmium-fixed neuroendocrine cells with a protein A-gold technique.

1987 ◽  
Vol 35 (7) ◽  
pp. 795-801 ◽  
Author(s):  
S A Hearn
Keyword(s):  
Neuroendocrine Tumors ◽  
Chromogranin A ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Protein A ◽  
Neuroendocrine Cells ◽  
Neurosecretory Granules ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Carotid Body Tumors

An antibody (LK2H10) to chromogranin A has been recommended for use in ultrastructural identification of neuroendocrine secretory granules. Previous studies have demonstrated immunoreactive chromogranin A in specimens prepared for electron microscopy by glutaraldehyde fixation only. In this study, the effect of specimen post-fixation by osmium tetroxide on post-embedding localization of chromogranin A was evaluated. Human tissues from benign endocrine glands, neuroendocrine tumors, and non-neuroendocrine tumors were post-fixed in osmium, embedded in epoxy resin, and the sample thin sections immunolabeled using a protein A-gold technique. Chromogranin A-positive neurosecretory granules were detected in pancreatic islets, adrenal medulla, stomach, ileum, anterior pituitary, and parathyroid. Mid-gut carcinoids, bronchial carcinoids, pheochromocytomas, paragangliomas, carotid body tumors, and thyroid medullary carcinomas contained immunoreactive granules. Cytoplasmic granules in non-neuroendocrine tumors did not react for chromogranin A. Tissues post-fixed in osmium tetroxide had optimally preserved ultrastructural features, and use of this fixative is compatible with postembedding localization of chromogranin A in neurosecretory granules.

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