M. Moeremans

High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method.

Abstract A new, simple procedure is described for the production of 5 nm colloidal gold/secondary antibody reagents. Utilizing them with antitubulin shows 1) that they can be used for high resolution ultrastructural localization studies and 2) that this can be done with retention of satisfactory preservation of cell structure. The same, simple procedure can be used to prepare 20 nm colloidal gold/antibody reagents. These can be used for the high resolution light microscopic visualization of microtubules in interphase and mitotic cells. Colloidal gold labelled serum or monoclonal antibodies can be used in a new, general purpose immunocytochemical technique: the IGS (immuno gold staining) method.

Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining

The use of colloidal gold and colloidal gold followed by silver enhancement as marker for dot or blot immune overlays is described. Colloidal gold probes concentrating at the sites of immune reaction gradually develop a pinkish colour during incubation that can be seen with the naked eye. With a physical developer, very high sensitivity and contrast is obtained by silver precipitation on the gold marker. Comparison of the immunogold and immunogold/silver staining methods with the indirect, PAP and ABC immunoperoxidase methods demonstrates that colloidal gold probes are excellent markers for immune overlay assays.

Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopy

We describe a new automatic technique for the study of intracellular mobility. It is based on the visualization of colloidal gold particles by video-enhanced contrast light microscopy (nanometer video microscopy) combined with modern tracking algorithms and image processing hardware. The approach can be used for determining the complete statistics of saltatory motility of a large number of individual moving markers. Complete distributions of jump time, jump velocity, stop time, and orientation can be generated. We also show that this method allows one to study the characteristics of random motion in the cytoplasm of living cells or on cell membranes. The concept is illustrated by two studies. First we present the motility of colloidal gold in an in vitro system of microtubules and a protein extract containing a kinesin-like factor. The algorithm is thoroughly tested by manual tracking of the videotapes. The second study involves the motion of gold particles microinjected in the cytoplasm of PTK-2 cells. Here the results are compared to a study using the spreading of colloidal gold particles after microinjection.

Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations.

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.

The Preparation of Colloidal Gold Probes and Their Use as Marker in Electron Microscopy

Cytochemical marking comprizes the visualization and localization of target molecules (targets) in cells and tissues. The targets are recognized by identifiers such as: antibodies (for antigens), lectins (for polysaccharides and glycoproteins), enzymes (for their substrate, e.g., polynucleotides, collagen, elastin, etc...), ligands (for their receptor or binding site), and derivatized polynucleotides (for in situ hybridization on isolated chromosomes and tissue sections).

Cytochemical profile of immunoregulatory T-lymphocyte subsets defined by monoclonal antibodies.

Immunogold staining in combination with enzyme cytochemistry was used to determine the cytochemical profile of the immunoregulatory T-lymphocyte subpopulations defined by the monoclonal antibodies OKT3, OKT4, OKT8, and OKM1 in normal peripheral blood. Leukocyte suspensions were first incubated with the monoclonal mouse antibodies and then with colloidal gold-labeled goat antimouse antibodies. The cells were fixed and cytocentrifuge preparations were made. Cytochemical reactions for the detection of peroxidase, acid alpha-naphthyl acetate esterase, acid phosphatase, and beta-glucuronidase were performed on these preparations. Under light microscopy, lymphocytes reacting with the monoclonal antibodies had numerous dark granules around their surface membrane. In the cytoplasm the intracellular enzymatic activities were stained. The T-lymphocytes were characterized by a dot-like activity for the three enzymes. No significant difference could be found between the cytochemical profile of the T-helper (OKT4 positive) and T-cytotoxic suppressor cell populations (OKT8 positive). A few cells with lymphocyte morphology reacted with the OKM1 monoclonal antibody. Their cytochemical characteristics were different from those of mature T-cells (OKT3 population) or mature monocytes. From the comparison of their cytochemical characteristics, we can conclude that there is little correlation between the immunoregulatory T-lymphocyte subsets defined by these monoclonal antibodies and those defined by Fc receptors.

The IGS (Immuno Gold Staining) Method used with Monoclonal Antibodies

Abstract A simple method is described for the production of stable and highly active monoclonal antibody/colloidal gold reagents. These reagents can be used in the recently introduced IGS (immuno gold staining) method to label antigens at both the light and electron microscopic level. As an example, the labelling of human T lymphocytes is given. It is concluded that the IGS method potentially lends itself very well for use with monoclonal antibodies.

Study of the parameters of binding of R 61837 to human rhinovirus 9 and immunobiochemical evidence of capsid-stabilizing activity of the compound.

The binding of the antiviral compound R 61837 to human rhinovirus 9 (HRV 9) was studied quantitatively and compared with binding of R 61837 to HRV 9H, a semiresistant variant. For both strains, radiolabelled R 61387 bound to native particles only. The Kd values obtained by Scatchard analysis of saturation binding data were 37 nM for HRV 9 and 172 nM for HRV 9H, whereas the concentrations resulting in a 50% reduction of cytopathic effect were 42 nM and 840 nM, respectively. Reversibility experiments showed that 65% of the compound could be extracted with chloroform from HRV 9H but less than 5% could be extracted from HRV 9. Dissociation studies demonstrated that in the presence of excess unlabelled compound, the half-lives of the virus compound complex HRV 9 and HRV 9H were 385 and 15 min, respectively. The effect of this antirhinoviral compound on the formation of subviral particles induced by low pH or heat was also investigated. Rate zonal centrifugation experiments using [35S]methionine-labelled HRV 9 showed that binding of R 61837 protected the virus against heat (56 degrees C) and acid (pH 5.0) and that at the same concentration of R 61837 the semiresistant strain was stabilized to a lesser extent. This observation was confirmed immunochemically with nonneutralizing and neutralizing monoclonal antibodies. Both 80S and 130S subviral particles have C antigenic determinants, whereas native particles (150S) have been designated D. R 61837 prevented the switch from D to C antigenicity which can be induced by exposure of rhinoviruses to mild denaturing conditions. These findings indicate that the compound is able to prevent a conformational change of the capsid which may be a prerequisite for infection. Images

Enumeration of T Lymphocytes and their Subclasses in Peripheral Blood by Immunogold Staining

Abstract Immunogold staining was used to enumerate T lymphocytes and their subclasses, as defined by monoclonal antibodies in the peripheral blood of a healthy population. Leukocytes were first incubated with the monoclonal antibodies and then with colloidal gold-labeled secondary antibodies. The cells were fixed and cytocentrifuge preparations were made. Granulocytes an monocytes in the preparations were labeled by cytochemical staining of their endogenous peroxidase activity. The preparations were examined in light microscopy and the percentage of positive lymphocytes was determined. The results were compared to those obtained by immunofluorescence microscopy. On stable preparations, lymphocytes could be recognized by their morphological characteristics. Cells reacting with the monoclonal antibody had numerous red-blue granules all around their surface membrane. The red-blue color of these granules is the color of the gold marker. This immunogold staining method forms a reliable tool for the enumeration of T lymphocytes and their subclasses. Applied on small quantities of whole blood, after lysis of the red blood cells, it could form an excellent routine procedure for this purpose, especially in laboratories lacking immunofluorescence equipment.

Ketanserin in Wound Healing and Fibrosis: Investigations into its Mechanism of Action

Ketanserin, an 5-HT2 receptor blocker, has a paradoxical effect in wound healing. It stimulates wound closure but inhibits hypertrophic scar formation. The drug stimulates proliferation and collagen synthesis of dermal fibroblasts but inhibits the 5-HT-induced DNA synthesis and retraction in myofibroblasts. Ketanserin is also bifunctional regulator of collagen synthesis in a sponge granuloma model. It increases the collagen content at day 4 but it lowers the collagen concentration in 14 day old granulomas. It is speculated that this differential response is due to a switch in serotonin receptor-type.