Light microscopical detection of leukocyte cell surface antigens with a one-nanometer gold probe

Summary The potential of ultrasmall gold particles for the light microscopical detection of leukocyte cell surface differentiation antigens was investigated. Suspensions and cytocentrifuge preparations of peripheral blood leukocytes were first incubated with monoclonal antibodies and then with goat antimouse antibodies coupled to colloidal gold particles of 1-nanometer diameter. Cytocentrifuge preparations were made from the cell suspensions. Silver enhancement was performed on all preparations. Then they were counterstained with May-Grünwald Giemsa and examined in light microscopy. The immunostaining appeared as fine dark granules on the surface membrane of the cells. Labeling conditions were determined which gave a dense specific immunostaining and a low background. High dilutions of the ultrasmall gold probe could be used to detect all antigen expressing cells in the samples. The labeling efficiency of the IGSS method with the 1 nanometer probe was comparable to that described earlier for 5 nanometer gold particles. Lymphocyte subsets enumerated with this method in normal peripheral blood were similar to those found with immunofluorescence microscopy. We concluded that one nanometer probes do not offer a major advantage in comparison with 5 nanometer probes for the study of cell surface antigens.     

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.     

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.     

An immunogold-silver staining method for detection of cell surface antigens in cell smears

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.     

Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.     

Colloidal gold probes in immunocytochemistry. An optimization of their application in light microscopy by use of silver intensification procedures

Colloidal gold particles are the markers of choice for ultrastructural localization of antigens. By reducing gold chloride with tannic acid and trisodium citrate, a broad range of narrowly determined particle sizes can be obtained. Such particles can easily be coupled to a number of proteins and the resulting conjugates are conveniently purified on a gel-chromatography column. Their application in light microscopy requires an amplification step with a silver physical developer. Silver-intensified colloidal gold probes can advantageously be used for immunostaining of cryostat, paraffin and plastic sections. Moreover, permeabilized cultured cells and whole-mount preparations can also be stained with gold-silver techniques. Silver intensification does not affect reactivity of a number of tissue antigens, thus permitting double staining combinations with immunoperoxidase or immunofluorescence methods.     

A multiple-staining ultrastructural procedure simultaneously detecting three membrane antigens on suspended cells by monoclonal antibodies and preembedding immunogold labelling

Summary A triple ultrastructural immunogold staining method for the simultaneous demonstration of three surface antigens of peripheral blood mononuclear cells at the electron microscope level is described. A six-step pre-embedding immunoelectron microscopy procedure was developed, using commercially available reagents. The CD11b antigen was first detected, through a two-step (indirect) method with 40 nm-sized gold particles; after a blocking step, the HLA-DR surface antigen was subsequently detected, through a two-step (biotin-streptavidin) method with 20 nm-sized gold particles; the CD4 antigen was finally detected, through a one-step (direct) method, using 5 nm-sized gold particles. Electron microscopic examination revealed firstly the presence of a triple-labelled cell subpopulation, which showed gold granules of the three sizes simultaneously decorating the cell membrane. Thus, the cells of such a subset simultaneously expressed the three antigens investigated. In contrast, either gold particles of only one size or no gold particles were observed on the cell surface of other subpopulations. This technique is a model demonstrating the importance of varying the size of particles in pre-embedding gold immunoelectron microscopy for a better analysis of the expression of surface antigens in isolated cells.     

Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.     

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.     

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35–1.0 m) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 m. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.     

Influence of biologically inspired nanometer surface roughness on antigen-antibody interactions for immunoassay-biosensor applications

Current research efforts to improve immunoassay-biosensor functionality have centered on detection through the optimal design of microfluidic chambers, electrical circuitry, optical sensing elements, and so on. To date, little attention has been paid to the immunoassay-biosensor membrane surface on which interactions between antibodies and antigens must occur. For this reason, the objective of the present study was to manipulate the nanometer surface roughness of a model immunoassay-biosensor membrane to determine its role on sensitivity and specificity. It was hypothesized that surface roughness characteristics similar to those used by the body's own immune system with B-lymphocyte cell membranes would promote antigen-antibody interactions and minimize non-specific binding. To test this hypothesis, polystyrene 96-well plate surfaces were modified to possess similar topographies as those of B-lymphocyte cell membranes. This was accomplished by immobilizing Protein A conjugated gold particles and Protein A conjugated polystyrene particles ranging in sizes from 40 to 860 nm to the bottom of polystyrene wells. Atomic force microscopy results provided evidence of well-dispersed immunoassay-biosensor surfaces for all particles tested with high degrees of biologically inspired nanometer roughness. Testing the functionality of these immunosurfaces using antigenic fluorescent microspheres showed that specific antigen capture increased with greater nanometer surface roughness while nonspecific antigen capture did not correlate with surface roughness. In this manner, results from this study suggest that large degrees of biologically inspired nanometer surface roughness not only increases the amount of immobilized antibodies onto the immunosurface membrane, but it also enhances the functionality of those antibodies for optimal antigen capture, criteria critical for improving immunoassay-biosensor sensitivity and specificity.     

Ultrastructural localization of the circulating anodic antigen and the circulating cathodic antigen in the liver of mice infected with Schistosoma mansoni: a sequential study

The present study describes the ultrastructural localization of two important circulating schistosome antigens--the circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA)--in livers of mice at various time intervals after infection with Schistosoma mansoni. For the demonstration of these antigens at the electron microscope level use was made of a direct, double immunogold labeling procedure, in which CAA-specific monoclonal antibodies, labeled with 5-nm gold particles, and CCA-specific monoclonal antibodies, labeled with 15-nm gold particles, were used. Both antigens were localized in granules and in inclusion bodies of Kupffer cells and granuloma macrophages and it was found that in these compartments the degree of 5- and 15-nm gold labeling increased with the duration of the infection. Sometimes gold particles were also encountered on the cell surface and in endocytotic vesicles of these cells, in endothelial cells, and in the space of Disse. From these data it was concluded that in the liver CAA and CCA were primarily accumulated in granules and inclusion bodies of Kupffer cells and granuloma macrophages. It is discussed whether at these locations both antigens are degraded by lysosomal enzymes and whether these antigens are complexed with antibodies.     

Silver enhancement of protein A-gold probes on resin-embedded ultrathin sections. An electron microscopic localization of mouse mammary tumor virus (MMTV) antigens

A simple method is described allowing the enhancement of the visibility of small gold probes for the electron microscopy. This method, which allows the silver intensification of gold directly on epon-embedded ultrathin sections, was used for the electron microscopic localization of Mouse Mammary Tumor Virus (MMTV) antigens in cultured cells derived from GR and BALB/cfRIII mouse mammary tumors. After the immunostaining with the preembedding protein A-gold technique, the ultrathin sections, placed on 200 mesh copper grids, were rehydrated and exposed to a photographic developer containing silver nitrate. During this physical development gold particles are incapsulated in growing shells of metallic silver, which gradually become more and more visible. We were able to obtain a heavy labelling of the viral particles, well visible even at low magnification, with a negligeable background staining. The present technique can be useful whenever it is necessary to use the smallest gold probes today available.     

Gold probe choice in simultaneous detection of human lymphocyte surface antigens at the ultrastructural level

A double immunogold-labeling method in immunoelectron microscopy was used for simultaneous detection of two antigens by monoclonal antibodies [OKT 8 (CD 8), anti-Leu-7, anti-Leu-11b (CD 16)] on lymphocytes in suspension. The combination of gold probe size (5 nm and 15 nm) and monoclonal antibody was found to be decisive for detecting double-labeled cells with the OKT 8+, Leu-11b+ phenotype. The combinations of OKT 8 labeled with the 5-nm gold probe (OKT 8(5] and anti-Leu-11b with the 15-nm gold probe (Leu-11b15) gave double-labeled cells; the reverse situation, using OKT 8 with a 15-nm gold probe (OKT 8(15] and anti-Leu-11b with a 5-nm gold probe (Leu-11b5), did not. Double-labeled OKT 8+, Leu-7+ cells were detected irrespective of which gold probe combination was applied. Our findings indicate that although the double immunogold-labeling method is well suited for study of lymphocyte subsets, it is important to determine suitable combinations of gold probe sizes and monoclonal antibodies for the lymphocyte subset under study, taking into account surface antigen density, so that double labeling ensues.     

Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography

The localization of scarce antigens in thin sections of biological material can be accomplished by pre-embedment labeling with ultrasmall immuno-gold labels. Moreover, with this method, labeling is not restricted to the section surface but occurs throughout the section volume. Thus, when combined with electron tomography, antigens can be localized in three dimensions in relation to the 3D (three-dimensional) ultrastructure of the cell. However, for visualization in a transmission electron microscope, these labels need to be enlarged by silver or gold enhancement. The increase in particle size reduces the resolution of the antigen detection and the large particles obscure ultrastructural details in the tomogram. In this paper we show for the first time that these problems can be avoided and that ultrasmall gold labels can be localized in three dimensions without the need for gold or silver enhancement by using HAADF-STEM (high angular annular dark-field-scanning transmission electron microscopy) tomography. This method allowed us to three-dimensionally localize Aurion ultrasmall goat anti-rabbit immuno-gold labels on sections of Epon-embedded, osmium-uranium-lead-stained biological material. Calculations show that a 3D reconstruction obtained from HAADF-STEM projection images can be spatially aligned to one obtained from transmission electron microscopy (TEM) projections with subpixel accuracy. We conclude that it is possible to combine the high-fidelity structural information of TEM tomograms with the ultrasmall label localization ability of HAADF-STEM tomograms.     

Labeling of binding sites for beta 2-microglobulin (beta 2m) on nonfibrillar surface structures of mutans streptococci by immunogold and beta 2m-gold electron microscopy.

As little detail is known about the surface structure of streptococci in the mutans group and the relationship of surface structure to host ligand-binding functions, the twofold purpose of this investigation was to examine in detail, by a range of electron microscopic techniques, the surface structures of streptococci in the different species of the mutans group and to investigate the distribution of beta 2-microglobulin (beta 2m)-binding sites on such structures. Strains representing Streptococcus mutans, S. cricetus, S. rattus, S. sobrinus, and four fresh isolates were studied by shadowcasting and histochemical staining of whole-mounted cells as well as by ultrathin and thick sectioning of embedded specimens. beta 2m-binding site distribution was visualized by indirect immunogold electron microscopy and by direct bacterial binding of beta 2m-conjugated gold probes. Shadowcast preparations revealed binding of gold probes to the cell surface of known beta 2m-binding strains but not to their polar fibrillar appendages. These long fibrils, common to all strains, were trypsin and sonication sensitive and stained with lead citrate but not with uranyl acetate or ruthenium red. More gold particles were bound by the indirect technique. For grid-mounted bacteria, the gold was mostly bound in clusters at the periphery of the cells. When gold probes were reacted in suspension with bacteria before mounting onto grids, a more even distribution of the gold was seen, but the bacteria were aggregated. Heating the bacteria eliminated beta 2m-gold binding but had no effect on the morphology of the fibrils. Thick sections of embedded bacteria prereacted with beta 2m-conjugated gold probes were analyzed by stereo imaging. A wispy, uranyl acetate-stained fuzzy layer, distinct from the fibrils seen by shadowcasting and extending up to one cell diameter from the cell wall, contained the gold probes. These findings introduce a concept that binding sites for some salivary ligands on mutans streptococci may be clustered on very delicate, nonfibrillar structures extending much further from the cell wall than previously appreciated. As for beta 2m, which composes part of the human histocompatibility antigens, part of the bacterial surface would be coated at a distance from its body with a protein not necessarily recognized as foreign by the host.     

A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level.

Pre-embedding double immunogold-silver labeling using two ultrasmall gold conjugates has not been attempted previously because a means of distinguishing labels by conjugates of identical sizes was lacking. This study investigated the feasibility of creating a particle size segregation between two ultrasmall gold conjugates through sequential immunogold incubations and silver enhancements. Two primary antibodies, mouse anti-synaptophysin and rabbit anti-glial fibrillary acidic protein (GFAP), were used in the model system. Differentiation of the double labeling was achieved by incubating with one ultrasmall gold conjugate, followed by silver enhancement, and then incubating with the second ultrasmall gold conjugate, followed by additional silver enhancement. This resulted in two groups of silver-enhanced particles: smaller particles enhanced once and larger particles enhanced twice. Electron microscopic examination revealed two readily distinguished populations of gold-silver particles within the appropriate structures, with very little size overlap. The quality of the ultrastructure permitted identification of most subcellular organelles. This procedure provides for the first time a pre-embedding immunogold-silver labeling protocol that allows the precise subcellular co-localization of multiple antigens.     

Fluorescence in situ hybridisation coupled to ultra small immunogold detection to identify prokaryotic cells using transmission and scanning electron microscopy

We describe a method based on fluorescence in situ hybridisation (FISH) that allows the identification of individual cells by electron microscopy. We hybridised universal and specific fluorescein-labelled oligonucleotide probes to the ribosomal RNA of prokaryotic microorganisms in heterogeneous cell mixtures. We then used antibodies against fluorescein coupled to sub-nanometer gold particles to label the hybridised probes in the ribosome. After increasing the diameter of the metal particles by silver enhancement, the specific gold-silver signal was visualised by optical microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is the first time that SEM is applied to the detection of gold nanoparticles hybridised to an intracellular target, such as the ribosome. The possibility to couple phylogenetic identification by FISH to cell surface and ultrastructure observation at electron microscopy resolution has promising potential applications in microbial ecology.     

Correlative light and electron microscopic immunocytochemistry on the same section with colloidal gold

Ultrastructural localization of growth hormone in rat anterior pituitary and of muscle-specific actin in rabbit arterial smooth muscle cells was accomplished with a post-embedment procedure using colloidal gold. Plastic sections (2 microns) were mounted on slides, deplasticized, immunostained with immunoglobulin-colloidal gold particles, re-embedded in Epon, and sectioned for electron microscopy. This procedure enabled light and electron microscopic localization of these intracellular antigens on the same section. Positive immunostaining was demonstrated with this procedure with a muscle-specific actin antibody which previously failed to localize antigenic sites by EM. The procedure described yielded staining of high specificity, with minimal background and well-preserved ultrastructure. This re-embedding technique is useful in situations where problems with post-embedding EM immunostaining exist and where correlative LM and EM immunostaining is essential.