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Biomedical subjects

J De Mey

Publications and source records attributed to J De Mey.

At least 37 records · Page 2Linked to original sources
PubMed1986

Sequential immunostaining (gold/silver) and complete protein staining (AuroDye) on Western blots.

A double staining method is described which combines immunodetection with sensitive staining of the complete electropherogram on the same membrane. The method is based on the use of Tween 20 as blocking agent, and uses immunogold/silver staining of specific antigens and gold staining of the overall protein pattern with AuroDye. This double staining makes possible the exact location of an immunodetected band within a complex protein pattern.

Animals↗
PubMed1986

FerriDye: colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes.

A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD460 nm = 0.5, followed by Perls' reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

Animals↗
PubMed1986

The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell.

We describe a new approach to probe the molecular biology of the living cell that uses small colloidal gold particles coupled to specific ligands. They are visualized in cells by bright-field, video enhanced contrast microscopy. We describe the basic aspects of the technique and provide examples of applications to intracellular motility, cell membrane dynamics, receptor translocation, internalization, and intracellular routing. We also provide examples of the use of this approach in immunospecific labelling of cells and tissue sections.

Animals↗
PubMed1986

Caldesmon is an elongated, flexible molecule localized in the actomyosin domains of smooth muscle.

A rapid purification procedure has been developed for the isolation of caldesmon from hog stomach smooth muscle utilizing a KI extract of washed myofibrils as source material. On SDS-PAGE this mammalian caldesmon showed a closely-spaced doublet around 155 kd. By low-angle rotary shadowing caldesmon was shown to be an elongated, highly flexible molecule which tends to form end-to-end dimers that are structurally very similar to filamin. When added to F-actin solutions caldesmon increased the high-shear viscosity considerably, but by an extent that depended on sample preparation. The effect was shown to be due to caldesmon and not to a trace contaminant by its full reversibility after addition of a monospecific caldesmon antibody. Recent investigations have shown that in smooth muscle two structurally distinct domains can be distinguished: an actomyosin domain and an actin-intermediate filament domain. Immunocytochemistry of ultrathin sections of smooth muscle at the light and electron microscope level revealed that caldesmon is present in the actomyosin domain. Caldesmon is thus a potential regulator of the actomyosin system in smooth muscle.

Actins↗
PubMed1986

The molecular organization of myosin in stress fibers of cultured cells.

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

Actin Cytoskeleton↗
PubMed1986

Localization of filamin in smooth muscle.

The distribution of contractile and cytoskeletal proteins in smooth muscle has been mapped by immunocytochemical methods, with special reference to the localization of the actin-binding protein, filamin. Immunolabeling of ultrathin sections of polyvinylalcohol-embedded smooth muscle distinguished two domains in the smooth muscle cell: (a) actomyosin domains, made up of continuous longitudinal arrays of actin and myosin filaments, and (b) longitudinal, fibrillar, intermediate filament domains, free of myosin but containing actin and alpha-actinin-rich dense bodies. Filamin was found to be localized specifically in the latter intermediate filament-actin domains, but was excluded from the core of the dense bodies. Filamin was also localized close to the cell border at the inner surface of the plasmalemma-associated plaques. In isolated cells the surface filamin label showed a rib-like distribution similar to that displayed by vinculin. It is speculated that the two domains distinguished in these studies may reflect the existence of two functionally distinct systems: an actomyosin system required for contraction and an intermediate filament-actin system, with associated gelation proteins, that is responsible, at least in part, for the slow relaxation and tone peculiar to smooth muscle.

Animals↗
PubMed1986

Detection of cell surface antigens in cryostat sections with immunogold-silver staining.

Immunogold-silver staining was used for the detection of lymphocyte cell surface antigens in cryostat sections of lymphoid tissues. The sections were incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained, and mounted. In light microscopy, the tissue architecture was well preserved, and a dark labeling was seen on the positive cells. Optimal labeling conditions were determined. The distribution of the lymphocyte subsets, as defined by a panel of monoclonal antibodies in tonsil and reactive lymph nodes, was similar to that found with a biotin-avidin-horseradish peroxidase method. The monoclonality of the neoplastic cells in lymph nodes of B-cell non-Hodgkin's lymphomas clearly could be demonstrated. The sensitivity of the technic was comparable with that of the biotin-avidin-horseradish peroxidase labeling method. In addition, immunogold-silver labeling was combined with acid phosphatase cytochemistry.

Antibodies, Monoclonal↗
PubMed1986

Immunogold-silver staining of lymphocyte surface antigens on cells in suspension and in lymph node cryostat sections.

An immunogold-silver staining (IGSS) technique for the light microscopical detection of leucocyte cell surface antigens in cell suspensions and cryostat sections is described. The specimens were first incubated with monoclonal mouse antibodies and then with colloidal gold-labelled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained and mounted. In light microscopy, the tissue architecture and the cellular morphology were well preserved. Positive cells showed dark granules on their surface membranes. Optimal labelling conditions were determined. This method proved to be a reliable tool for the enumeration of T-cells and their subsets in peripheral blood. The dense labelling permitted the use of panoptic counterstains like May-Grünwald-Giemsa or Wright's stain. This IGSS technique was used to determine the distribution of the T- and B-cell subsets in cryostat sections of reactive lymph nodes. The sensitivity of the method was comparable with that of immunofluorescence microscopy for cell suspensions and that of the biotin-avidin-peroxidase technique for tissue sections. Immunogold-silver staining was combined with enzyme cytochemistry. In dark-field or epipolarization microscopy the labelling appeared as bright granules on a dark background. With its dense granular membrane labelling and its good morphology IGSS is an ideal method for the study of particular cell types in mixed cell suspensions. In addition, it could be a general method for the detection of cell surface antigens in all kinds of cells and tissues.

Antigens, Surface↗
PubMed1986

Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibodies.

The potential of immunogold-silver staining for study of leukocyte subpopulations, as defined by monoclonal antibodies in cell suspensions, was examined. The cells were labeled in suspension as described for immunogold staining. Cytocentrifuge preparations of the suspensions were then immersed in a physical developer. By light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. The morphology of the cells, as revealed by a panoptic counterstain, was comparable with that seen in routine cell smears for differential counts. The numbers of T-cells, T-helper/inducer cells, and T-cytotoxic/suppressor cells counted by this method in normal peripheral blood were nearly identical to those identified by immunogold staining and immunofluorescence microscopy in the same cell suspensions. The good morphological delineation also made possible rapid and accurate identification of particular leukocyte subsets in complex cell suspensions. Atypical lymphocytes from patients with infectious mononucleosis displayed the surface phenotype of activated T-cytotoxic/suppressor cells. Different maturation stages of neoplastic cells in patients with acute myeloid leukemia showed differences in surface antigen expression. Immunological detection of cell surface antigens could be combined with cytochemical staining of intracellular enzymatic activities. Finally, the labeling could be performed on cells prefixed on glass slides.

Adult↗
PubMed1986

An immunogold-silver staining method for detection of cell-surface antigens in light microscopy.

An immunogold-silver staining technique for detection of cell-surface antigens in cell suspensions was developed. Leukocyte cell suspensions were first incubated with monoclonal antibodies directed against cell-surface antigens and then with colloidal gold-labeled goat anti-mouse antibodies. Cytocentrifuge preparations of the cell suspensions were immersed in a physical developer containing silver lactate and hydroquinone as reducing substance. The preparations were then counterstained and mounted. In light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. An optimal morphology, as revealed by a May-Grünwald-Giemsa counterstain, permitted accurate cell identification. The labeling was influenced by the gold particle diameter and the concentration of the gold reagents, by the duration of incubation in the physical developer, and by the composition and temperature of this medium. The T-cell subsets enumerated with this method in the peripheral blood of normal adults were identical to those found with other methods. The sensitivity of the technique was comparable with that of immunofluorescence microscopy. This immunogold-silver staining procedure proved to be a reliable tool for detection of cell-surface antigens in light microscopy.

Antibodies, Monoclonal↗
PubMed1985

Localization of somatostatin-, bombesin-, and serotonin-like immunoreactivity in the lung of the fetal rhesus monkey.

Immunoreactivity of regulatory peptides has been demonstrated in the fetal lung of Macaca mulatta by the peroxidase anti-peroxidase method. Serotonin-immunoreactive neuroepithelial bodies are distributed in the airways from the bronchi to the alveolar ducts. Many neuroepithelial bodies also show bombesin-like immunoreactivity; a very few are immunoreactive to somatostatin antiserum. Four populations of neuroepithelial bodies were identified which contain immunoreactivity for 1) serotonin alone, 2) serotonin and bombesin, 3) serotonin and somatostatin, and 4) serotonin, bombesin, and somatostatin. Since bombesin and somatostatin have been demonstrated to have opposite effects on the release of other peptide hormones, it seems likely that the presence of these same peptides in neuroepithelial bodies may have a similar regulatory role in the lung.

Animals↗
PubMed1985

Increased numbers of neuroepithelial bodies (NEB) in lungs of fetal rhesus monkeys following maternal dexamethasone treatment.

Numbers of neuroepithelial bodies in the lungs of fetal Rhesus monkeys increase in a dose-dependent manner when mothers are treated antenatally with dexamethasone. Maternal doses of 15 mg/kg result in numbers of neuroepithelial bodies in lungs of fetuses of 135-day gestational age not different from those of the mature control group at 162 days. Controls at 135 days are significantly different from those at 162 days. Serotonin immunoreactive neuroepithelial bodies were localized by immunocytochemistry.

Animals↗
PubMed1985

Immuno gold staining (IGS) and immuno gold silver staining (IGSS) for the identification of the plant pathogenic bacterium Erwinia amylovora (Burrill) Winslow et al.

For the identification of the plant pathogenic bacterium Erwinia amylovora, the immuno gold staining (IGS) and immuno gold silver staining (IGSS) techniques are tested. The IGS and IGSS methods are at least as sensitive an indirect immunofluorescence and require less primary antiserum. Moreover they have the advantage that the preparations can be conserved permanently and unchanged. The preparation of the IGS can be observed with transmitted light or--with considerable better result--using epipolarization microscopy. The IGSS method deserves special attention because of its high contrast in normal brigth field microscopy with transmitted light.

Animals↗
PubMed1985

Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes.

A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

Animals↗