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.

scholarly journals Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.

1991 ◽  
Vol 39 (9) ◽  
pp. 1267-1279 ◽  
Author(s):  
M van Lookeren Campagne ◽  
A B Oestreicher ◽  
T P van der Krift ◽  
W H Gispen ◽  
A J Verkleij
Keyword(s):  
Monoclonal Antibodies ◽  
Rat Brain ◽  
Anhydrous Methanol ◽  
Protein A ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Polyclonal Antiserum ◽  
Double Labeling ◽  
Ultrastructural Studies

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.

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 the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies.

1984 ◽  
Vol 98 (6) ◽  
pp. 2239-2244 ◽  
Author(s):  
R Sealock ◽  
B E Wray ◽  
S C Froehner
Keyword(s):  
Monoclonal Antibodies ◽  
Acetylcholine Receptor ◽  
Protein A ◽  
Spatial Relationship ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Postsynaptic Membrane ◽  
Specific Protein ◽  
Cytoplasmic Surface ◽  
Membrane Bound

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.

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.

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.

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.

scholarly journals Quick-freezing and freeze-drying in preparation for high quality morphology and immunocytochemistry at the ultrastructural level: application to pancreatic beta cell.

1982 ◽  
Vol 30 (2) ◽  
pp. 129-138 ◽  
Author(s):  
R W Dudek ◽  
G V Childs ◽  
A F Boyne
Keyword(s):  
Beta Cell ◽  
Secretory Granules ◽  
Pancreatic Beta Cell ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Freeze Dried ◽  
Quick Freezing ◽  
Degree Of Confidence ◽  
Freeze Fixation ◽  
Unlabeled Antibody

Quick-freeze fixation and freeze-dry methods were used successfully to obtain ultrastructural localization of insulin in the pancreatic beta cell by the unlabeled antibody-enzyme technique. In unosmicated freeze-fixed and freeze-dried islets, insulin was specifically demonstrated over the dense core of the secretory granules. In osmicated freeze-fixed and freeze-dried islets, insulin antigenicity withstood the osmium tetroxide vapor treatment. In addition, the surrounding ultrastructural resolution of morphologic features was significantly improved, which allowed insulin to be localized not only in secretory granules, but also in intracellular membranous compartments, with a degree of confidence not heretofore possible. Extracellular sites of insulin positivity in the islet were also localized and possible exocytotic activity for showing insulin release was observed.

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 lysozyme in human colon eosinophils using the protein A-gold technique: effects of processing on probe distribution.

1989 ◽  
Vol 37 (5) ◽  
pp. 709-714 ◽  
Author(s):  
J W Stirling
Keyword(s):  
Protein A ◽  
Acrylic Resin ◽  
Ultrastructural Localization ◽  
Human Colon ◽  
Similar Distribution ◽  
Sodium Metaperiodate ◽  
Specific Granules ◽  
White Tissue ◽  
The Matrix ◽  
Standard Techniques

The distribution of lysozyme (muramidase) within eosinophil leukocytes situated in the lamina propria of human colon was studied by immunoelectron microscopy using a range of standard techniques. Tissue processed in a variety of glutaraldehyde- or paraformaldehyde-based fixatives was partially dehydrated and embedded in the acrylic resin LR White. Tissue thus treated showed lysozyme in pale cytoplasmic granules and the matrix of specific granules, but not in their crystalloids. Trypsinization of sections had little effect on this result, and tissues fixed in glutaraldehyde and embedded in Araldite showed a low level of reactivity with a similar distribution. After etching LR or Araldite sections with sodium metaperiodate, the pale granules and specific granule matrices became negative for lysozyme and the crystalloids became positive. Because crystalloids also were labeled with normal rabbit immunoglobulin after etching, this apparent redistribution of label could be due to nonspecific binding rather than exposure of masked epitope.

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