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 A domain-specific marker for the hepatocyte plasma membrane. II. Ultrastructural localization of leucine aminopeptidase to the bile canalicular domain of isolated rat liver plasma membranes.

1984 ◽  
Vol 98 (4) ◽  
pp. 1488-1496 ◽  
Author(s):  
L M Roman ◽  
A L Hubbard
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Leucine Aminopeptidase ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Companion Paper ◽  
Specific Marker ◽  
Cell Biol ◽  
Affinity Isolation

Leucine aminopeptidase (LAP) is an integral membrane glycoprotein localized to the apical membrane domain of intestinal and kidney epithelial cells. By indirect immunofluorescence, we have shown that antibodies raised against rat intestinal LAP recognized a similar protein concentrated in the bile canalicular (BC) domain of the hepatocyte in situ (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558). We have extended this localization to the ultrastructural level. When a saponin-permeabilized, agarose-embedded plasma membrane (PM) fraction was incubated with affinity-purified anti-LAP, 85% of the protein A-gold particles associated with the three recognizable PM domains were present in the BC. The levels of labeling on the other two domains (sinusoidal and lateral) did not exceed that observed with nonimmune controls. The concentration of LAP in the BC domain in isolated PM sheets prompted us to use this antigen for the affinity isolation of BC membrane (Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1497-1504, companion paper).

Spreading of a sperm surface antigen within the plasma membrane of the egg after fertilization in the rat

Development ◽  
1983 ◽  
Vol 75 (1) ◽  
pp. 259-270
Author(s):  
Stephen J. Gaunt
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Guinea Pig ◽  
Surface Antigen ◽  
Plasma Membranes ◽  
Entire Surface ◽  
Sperm Tail ◽  
Sperm Surface ◽  
Sperm Antigens

The rat sperm surface antigen 2D6, located over the entire surface of the spermatozoon, is shown by use of a monoclonal antibody in indirect immunofluorescence experiments to spread laterally over the surface of the egg after fusion of sperm and egg plasma membranes at fertilization. Freshly fertilized eggs, obtained from superovulated rats 14h after hCG injection, showed the 2D6 antigen to have spread in a gradient over a discrete fan-shaped area of the egg surface anterior to the protruding sperm tail. Eggs at a later stage of sperm incorporation, obtained 20 h after hCG injection, snowed that the spread of antigen had extended to cover most or all of their surfaces. By 40 h after hCG injection, the approximate time that fertilized eggs cleaved to form 2-cell embryos, most of the 2D6 antigen had been lost from the cell surface. Fertilized eggs, but not unfertilized eggs or 2-cell embryos, were lysed by 2D6 monoclonal antibody in the presence of guinea pig complement. A model for sperm-egg fusion is presented to account for the observed pattern of spreading shown by the 2D6 antigen. The possible role of sperm antigens on the egg surface is discussed.

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.

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.

Characterisation of the progesterone receptor on canine spermatozoa

2005 ◽  
Vol 17 (7) ◽  
pp. 733 ◽  
Author(s):  
Jui-Te Wu ◽  
Pei-Shiue Tsai ◽  
Shuang-Lin Lee ◽  
Feng-Pang Cheng
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Progesterone Receptor ◽  
Seminal Plasma ◽  
High Capacity ◽  
Fluorescein Isothiocyanate ◽  
Dependent Manner ◽  
Sperm Plasma Membrane ◽  
Pre Treatment ◽  
Dose Dependent

The present study was conducted to characterise and localise the progesterone receptor (PR) on canine spermatozoa. Using a progesterone–bovine serum albumin–fluorescein isothiocyanate conjugate (PBF) and different monoclonal antibodies (C262 and NCL-PGR against the steroid binding domain and N-terminus of intracellular PR, respectively, and h151 against the hinge domain of the intracellular oestrogen receptor), the PR was identified on the plasma membrane over the acrosomal region. Two proteins (54 kDa and 65 kDa) were detected by recognition of the three monoclonal antibodies using Western blotting. PBF labelling was observed in the majority of cauda epididymal spermatozoa (63 ± 4%), but this labelling was markedly reduced (33 ± 17%) after the addition of canine seminal plasma. Over a 7-h capacitation, the proportion of ejaculated spermatozoa exhibiting PBF labelling (indicating the presence of the PR) increased from 18 ± 10% (onset) to 59 ± 7% by 5 h, where it plateaued. Progesterone (P4) induced the acrosome reaction (AR) in a dose-dependent manner (0, 0.1, 1 and 10 µg/mL P4 corresponding to 10 ± 5%, 16 ± 9%, 23 ± 7% and 30 ± 7%). Pre-treatment of capacitated spermatozoa with canine seminal plasma reduced the incidence of the P4-induced AR (12 ± 5%). In addition, treatment with the monoclonal antibodies significantly reduced the incidence of the P4-induced AR (10 µg/mL) in capacitated ejaculated spermatozoa from 19 ± 6% to 11 ± 4% (h151, 1 : 10) and 12 ± 6% (C262, 1 : 10), respectively. A typical Scatchard plot revealed one binding with high affinity and low capacity, and another binding with low affinity and high capacity, suggesting at least two different characteristic PR. Taken together, these results demonstrate that P4 induced the AR in a dose-dependent manner via functional transmembranal receptors in the acrosomal region of the canine sperm plasma membrane. The characteristics of this membrane receptor seem similar to those of other mammalian spermatozoa, and it shows structural homology to the intracellular PR.

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 Correlative immunofluorescence and electron microscopy on the same section of epon-embedded material.

1985 ◽  
Vol 33 (2) ◽  
pp. 165-171 ◽  
Author(s):  
C L Rieder ◽  
S S Bowser
Keyword(s):  
Electron Microscopy ◽  
Core Protein ◽  
Fluorescein Isothiocyanate ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Immunofluorescent Localization ◽  
Cellular Inclusions ◽  
Tumor Virus ◽  
Specific Antigens ◽  
First Time

Semithick (0.25-0.50 micron) sections, cut from cells stained with fluorescein isothiocyanate (FITC)-conjugated antibodies prior to embedding in Epon, show high resolution patterns of immunofluorescence against a background void of autofluorescence. These same sections can then be viewed, after uranyl and lead staining, in the electron microscope. We clearly establish the specificity of this same-section correlative immunofluorescence-electron microscopy approach by showing that the immunofluorescent patterns observed in such sections of cells, stained prior to embedding for the indirect immunofluorescent localization of tubulin, follows the distribution of microtubules within the same sections as determined by electron microscopy. We then use this method to demonstrate for the first time that the 57 kD core protein of wound tumor virus is associated, at the ultrastructural level, with two distinct cellular inclusions in virally infected AC-20 cells. In some instances the fidelity in the correlation between the distribution of immunofluorescently labeled antigens and the ultrastructure in the same section eliminates the need to employ more complex procedures for labeling antigens for ultrastructural detection. This technique, therefore, provides a rapid and simple first approach to many problems that require the ultrastructural localization of specific antigens.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF CHORIONIC GONADOTROPIN IN HUMAN TERM PLACENTA

1970 ◽  
Vol 18 (12) ◽  
pp. 862-874 ◽  
Author(s):  
R. B. DRESKIN ◽  
S. S. SPICER ◽  
W. B. GREENE
Keyword(s):  
Plasma Membrane ◽  
Chorionic Gonadotropin ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Dense Granule ◽  
Apical Plasma Membrane ◽  
Small Variety ◽  
Basal Plasma ◽  
Antigen Antibody ◽  
And Control

Human chorionic gonadotropin (HCG) was localized in human placenta at the ultrastructural level by an immunocytochemical method which linked the antigen HCG to a peroxidase label through the antigen-antibody reactions of an immunoglobulin-peroxidase bridge. Material on the maternal surface of the apical plasma membrane and in the cisternae of rough endoplasmic reticulum of the syncytiotrophoblast displayed strong immunostaining for HCG and the outer surface of the basal plasma membrane appeared equivocally immunoreactive. Lamellae and vesicles of the Golgi complex of the syncytium lacked electron-opaque reaction product indicative of immunostaining for HCG. Deposits of peroxidase reaction product in the large variety of cytoplasmic dense granule varied from negligible to abundant in immunostained and in control specimens alike and, accordingly, the large granules appeared to contain intrinsic peroxidase but not HCG. The question of the immunoreactivity of the small variety of cytoplasmic dense granule was not conclusively settled, but the occurrence of reactive and unreactive small granules in both immunostained and control specimens weighed against the presence of HCG in these rather infrequent granules. The cytotrophoblast lacked evidence of immunoreactivity.

Facts and artifacts in colloidal gold postembedding cytochemistry

1986 ◽  
Vol 44 ◽  
pp. 44-47
Author(s):  
Moise Rendayan
Keyword(s):  
Binding Sites ◽  
Colloidal Gold ◽  
Protein A ◽  
Light And Electron Microscopy ◽  
Ultrastructural Localization ◽  
Surface Antigens ◽  
Enzyme Cytochemistry ◽  
Intracellular Binding ◽  
Gold Marker

The colloidal gold marker was introduced in immunocytochemistry by Faulk and Taylor, in 1971, for the ultrastructural localization of surface antigens. Since then, application of this marker in light and electron microscopy has been growing rapidly. In particular, it has been applied for postembedding labeling of intracellular binding sites and its use has been extended to the various fields of cytochemistry: immunocytochemistry (protein A-gold), IgG-gold), enzyme-cytochemistry and lectincytochemistry. Several reviews have been recently published on colloidal gold labeling techniques and we refer to them for extensive characterization of this marker and its various applications.

Monoclonal antibodies which recognize equatorial segment epitopes presented de novo following the A23187-induced acrosome reaction of guinea pig sperm

1995 ◽  
Vol 108 (2) ◽  
pp. 767-777 ◽  
Author(s):  
C.A. Allen ◽  
D.P. Green
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
De Novo ◽  
Convex Surface ◽  
Fluorescein Isothiocyanate ◽  
Ultrastructural Level ◽  
Immunogold Labelling ◽  
Cytoplasmic Face

Acrosome-intact mammalian sperm can adhere to zona pellucida-free oocytes but are only capable of fusing if they have previously undergone the acrosome reaction. This suggests that the acrosome reaction results in presentation of at least one novel epitope which plays a role in sperm-oocyte fusion. Monoclonal antibodies were raised against unfixed acrosome-reacted guinea pig sperm and screened by indirect immunofluorescence for binding to the equatorial segment. They were back-screened against unfixed acrosome-intact sperm for absence of binding. Using this approach, two antibodies, G11 and M13, were identified which detect equatorial segment epitopes presented de novo by sperm following an A23187-induced acrosome reaction. The localization of these epitopes to the equatorial segment was confirmed at the ultrastructural level by indirect immunogold-labelling. Fluorescein isothiocyanate-labelled Fab fragments of these two antibodies also localized to the equatorial segment. Affinity chromatography and western blotting established that the two mAbs recognize the same proteins, which have M(r)s of 34, 46, 48 and 51 × 10(3). When sperm were induced to undergo the acrosome reaction with A23187 and incubated with their discharged acrosomal contents, a further band was produced with an M(r) of 30 × 10(3). Production of this band was inhibited in the combined presence of 100 microM phenylmethylsulphonyl fluoride and 100 microM p-aminobenzamidine even though these compounds do not inhibit acrosomal exocytosis. Neuraminidase and O-glycosidase were without effect on the proteins detected by antibodies G11 and M13. Endoglycosidase F, however, eliminated the bands of M(r) 46, 48 and 51 × 10(3) and replaced them with a strong band of M(r) 44 × 10(3) and two minor bands of M(r) 43 and 45 × 10(3). Formaldehyde fixation of acrosome-intact sperm caused partial rupture of the acrosome with loss of the characteristic rouleaux (stacks) of guinea pig sperm. Indirect labelling of these formaldehyde-fixed sperm with fluorescein isothiocyanate- or gold-labelled second antibody, with or without permeabilization with 0.05% Triton X-100, showed dense labelling on the cytoplasmic face of the plasma membrane overlying the convex surface of the acrosome but little labelling elsewhere. Cryosections of acrosome-intact sperm labelled indirectly with immuno-gold showed labelling consistent with the same location, as well as sporadic labelling at other intracellular sites overlying the acrosome. Since there is no evidence that sperm can translocate intact membrane protein from the cytoplasmic face to the extracellular face of the plasma membrane during the acrosome reaction, the evidence suggests that there are two isolated antigen pools.(ABSTRACT TRUNCATED AT 400 WORDS)

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