Detection of cell surface antigens in tissue sections by means of pre-embedding immunogold staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualize cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

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.     

Ultrastructural immunocytochemistry of glia cells. Double labeling studies using LR White embedding and colloidal gold

The introduction of acrylate resins (Lowicryl K4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class II antigens by pre-embedding method then followed by post-embedding IGS for the other intracytoplasmic antigens.     

Ultrastructural immunocytochemistry of glia cells

Summary The introduction of acrylate resins (Lowicryl K 4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class. H antigens by pre-embedding method then followed by post-embedding IGS for the other intracytopasmic antigens.The Clinical Research Unit for Multiple Sclerosis is supported by Hermann and Lilly Schilling foundation     

Immunohistochemical localization of cell surface receptors using a novel method permitting simple, rapid and reliable LM/EM correlation

Summary A method is presented which allows correlative serial section analysis by light and electron microscopy of cell surface antigens in monolayer cultures. Sites of antigenicity are shown by deposition of diaminobenzidine after pre-embedding, immunoperoxidase immunocytochemistry. Osmication is replaced by the use of gold chloride which specifically enhances the electron density of diaminobenzidine. In addition gold chloride bound to diaminobenzidine survives embedding and provides the basis for a post-embedding photochemical amplification method. Immunostained cells are embedded in LR White by a rapid technique which preserves their structure and leaves them available for subsequent post-embedding immunocytochemistry. The method is illustrated by the demonstration of epidermal growth factor (EGF) receptors on the EGF receptor-rich human carcinoma cell line A431 using a well characterized monoclonal antibody raised against EGF receptor.     

Double immunogold staining method for the simultaneous ultrastructural localization of regulatory peptides

Recent studies have suggested that the morphological characteristics of secretory granules contained within endocrine cells and nerves may be determined largely by their chemical composition. The use of the immunogold staining (IGS) method, which is based on the adsorption of colloidal gold to immunoglobulins, has been used in our laboratory to demonstrate a wide range of intracellular antigens at both the light and electron microscope levels. In this study we have applied a modification of the IGS method for the simultaneous detection of two separate antigens in a single tissue section, using a variety of region-specific antisera to different peptides. Peptide antisera were raised in rabbits or in guinea pigs and these were applied simultaneously or sequentially to grid-mounted ultrathin tissue sections. Antigenic sites were visualized at the electron microscope level using antisera raised in goats, adsorbed to gold particles of 12, 20, or 40 nm. Using this technique we have attempted to investigate the coexistence of multiple antigens in single tissue sections, in particular in single granules; the topographic distribution of molecular forms within one single granule or granule population; the heterogeneity of peptidergic neurons and also the heterogeneity of peptide content in morphologically similar granules. The double immunogold staining procedures described here have proved to be extremely effective for the simultaneous ultrastructural localization of two antigens (peptide-peptide; peptide-propeptide) on a single tissue section. The further development of this technique may provide useful information on neuroendocrine cell dynamics in normal and diseased states.     

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.     

蚕豆（Vicia faba L．）叶肉细胞中ABA的胶体金免疫电镜定位

利用胶体金免疫电镜定位技术对蚕豆叶肉细胞中ＡＢＡ定位的研究表明,在以ＡＢＡ抗体处理的切片中,叶绿体有大量的金颗粒标记,细胞质和细胞核也有金颗粒标记,但液泡和细胞壁中没有金颗粒标记。免疫染色前用胰蛋白酶处理可显著增强金颗粒标记密度,而不用ＥＤＣ固定或以免疫前兔血清处理的切片中几乎没有金颗粒标记。本实验为蚕豆叶肉细胞中ＡＢＡ的分布提供了直接的证据并说明了该技术是研究ＡＢＡ定位的一种可靠的方法。     

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.     

Introduction of tyramide signal amplification (TSA) to pre-embedding nanogold-silver staining at the electron microscopic level.

The tyramide signal amplification (TSA) technique has been shown to detect scarce tissue antigens in light and electron microscopy. In this study we applied the TSA technique at the electron microscopic level to pre-embedding immunocytochemistry. This protocol was compared to the non-amplified protocol. With the TSA protocol, the labeling of GM130, a cis-Golgi matrix protein, was tested in a cell line and found to be highly sensitive and more enhanced than that with the simple protocol. Moreover, the gold particles were well localized to the cis-side of the Golgi apparatus in both the TSA and the simple protocol.     

The silver anniversary of gold: 25 years of the colloidal gold marker system for immunocytochemistry and histochemistry

Since 1971, when W.P. Faulk and G.M. Taylor published “An immunocolloid method for the electron microscope”, colloidal gold has become a very widely used marker in microscopy. It has been used to detect a huge range of cellular and extracellular constituents by in situ hybridization, immunogold, lectin-gold, and enzyme-gold labeling. Besides its use in light microscopic immunogold and lectin-gold silver staining, colloidal gold remains the label of choice for transmission electron microscopy studying thin sections, freeze-etch, and surface replicas, as well as for scanning electron microscopy. The year 1996 is the 25th anniversary of the introduction of colloidal gold as a marker in immunoelectron microscopy and this overview outlines some of the major milestones in the development of the colloidal gold marker system.     

Immuno-light and immuno-electron microscopic examination of freeze-dried kidney tissue

Correlative morphology of kidney tissue can be accomplished by a combination of immunofluorescent staining and light microscopy using a pre-embedding technique. Further development of this method has been elaborated by freeze-drying and chopping of the tissue, which then is incubated in biotinylated immunoglobulins. Fluorochromes and colloidal gold can be coupled to the biotinylated immunoglobulins in a second step. This technique is an alternative to postembedding immunogold staining for immunoelectron microscopy, and permits a correlative immunohistochemical and detailed morphological study of kidney tissue.     

Immuno-Light and Immuno-Electron Microscopic Examination of Freeze-Dried Kidney Tissue

Correlative morphology of kidney tissue can be accomplished by a combination of immunofluorescent staining and light microscopy using a pre-embedding technique. Further development of this method has been elaborated by freeze-drying and chopping of the tissue, which then is incubated in biotinylated immunoglobulins. Fluorochromes and colloidal gold can be coupled to the biotinylated immunoglobulins in a second step. This technique is an alternative to postembedding immunogold staining for immunoelectron microscopy, and permits a correlative immunohistochemical and detailed morphological study of kidney tissue.     

Physics of a rapid CD4 lymphocyte count with colloidal gold

The inherent surface charges and small diameters that confer colloidal stability to gold particle conjugates (immunogold) are detrimental to rapid cell surface labeling and distinct cluster definition in flow cytometric light scatter assays. Although the inherent immunogold surface charge prevents self aggregation when stored in liquid suspension, it also slows binding to cells to timeframes of hours and inhibits cell surface coverage. Although the small diameter of immunogold particles prevents settling when in liquid suspension, small particles have small light scattering cross sections and weak light scatter signals. We report a new, small particle lyophilized immunogold reagent that maintains activity after 42°C storage for a year and can be rapidly dissolved into stable liquid suspension for use in labelling cells with larger particle aggregates that have enhanced scattering cross section. Labeling requires less than 1 min at 20°C, which is ～30 times faster than customary fluorescent antibody labeling. The labeling step involves neutralizing the surface charge of immunogold and creating specifically bound aggregates of gold on the cell surface. This process provides distinct side-scatter cluster separation with blue laser light at 488 nm, which is further improved by using red laser light at 640 nm. Similar comparisons using LED light sources showed less improvement with red light, thereby indicating that coherent light scatter is of significance in enhancing side-scatter cluster separation. The physical principles elucidated here for this technique are compatible with most flow cytometers; however, future studies of its clinical efficacy should be of primary interest in point-of-care applications where robust reagents and rapid results are important.     

Counting Chromosomes in Root Tips from a Large Population of Plants

Correlative morphology of kidney tissue can be accomplished by a combination of immunofluorescent staining and light microscopy using a pre-embedding technique. Further development of this method has been elaborated by freeze-drying and chopping of the tissue, which then is incubated in biotinylated immunoglobulins. Fluorochromes and colloidal gold can be coupled to the biotinylated immunoglobulins in a second step. This technique is an alternative to postembedding immunogold staining for immunoelectron microscopy, and permits a correlative immunohistochemical and detailed morphological study of kidney tissue.     

Quantitative immunolocalization of four immunodominant antigens of Toxoplasma gondii

Ultrastructural localization of four immunodominant antigens of Toxoplasma gondii was investigated quantitatively on thin sections and replicas by an immunogold technique using four monoclonal antibodies (Mab). On immunoblot Mab IV47, GII9, II38 and IE10 identified proteins of 28, 30, 45 and 66-70 kDa, respectively. Use of digital image analyzer and a semi-automatic procedure developed by us, the patterns of label distribution were compared in three cell structures: cell surface, submembrane area and rhoptries. On the whole cell surface, protein P28 and P30 were 2.5 and 4 times more abundant than P66-70 respectively. The protein P28 was essentially concentrated in the submembrane area with a labeling of 195.4 +/- 46.7 gold particles/microns 2 that follows a decreasing gradient from this area to the cell centre. In the rhoptries, all four antigens were detected, P45 and P66-70 being major with a labeling of 97.1 +/- 31.1 gold particles/microns 2 and 155.1 +/- 39.3 gold particles/microns 2 respectively. The results support the hypothesis that rhoptries are the essential site for antigen storage.     

Localization and quantification of hormones, ligands, and mRNA with affinity-gold probes

The application of immunogold techniques to localize pituitary hormones produces label that can be quantified and correlated with different secretory states. This report focuses on three major applications of the technology. In the first set of studies, immunogold labels for adrenocorticotropin (ACTH) or luteinizing hormone (LH beta) and follicle-stimulating hormone (FSH beta) were applied to ultrathin sections of pituitaries from adrenalectomized rats or from rats in different stages of the estrous cycle. During the first week after adrenalectomy, ACTH cell area increased. The concentration of immunoperoxidase label (amount of label/area of the corticotropes) decreased. Counts of gold markers showed that there were no changes in the concentration of antigens per granule. Three weeks after adrenalectomy, the amount of immunoperoxidase label increased along with the concentration of that label. The concentration of gold label for ACTH on granules also increased. All changes correlated well with increases in serum ACTH stimulated by adrenalectomy. In the studies of cycling rats, gonadotropes showed increases in the number of gold markers for LH beta or FSH beta per granule area just before an elevation in serum levels. There were also increases in the proportion of granules that contained only LH beta or FSH beta (monohormonal) before the rise in secretion. Thus, nonparallel release of gonadotropins might be attributed to changes in the ratio of gonadotropins packaged per granule. In the second series of studies, avidin-gold labels were used to identify sites of binding of biotinylated ligands. These studies illustrate and quantify binding by biotinylated gonadotropin-releasing hormone (GnRH) to ovarian or pituitary target cells. Triple-labeling protocols (avidin-peroxidase followed by immunogold) show that the target cells in the pituitary contain gonadotropins. In the third set of studies, avidin gold or avidin peroxidase was used to label sites of hybridization of a biotinylated cRNA probe to gonadotropin beta subunit mRNA. The sites of hybridization appear on rough endoplasmic reticulum; however, further work is needed to improve cell ultrastructure and perserve antigens. Triple-labeling protocols (avidin-peroxidase followed by immunogold) show the feasibility of the technique as well as the need for further refinement. To summarize, these studies describe multiple applications of gold labels for the localization of antigens, ligands, and mRNA. The labels are sensitive for detection of antigens and ligands and easily quantified. Quantitative analyses show changes in concentration of gold label that correlate well with secretory states.     

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.     

Purification and identification of murine epidermal dendritic cells: flow cytometry and immunogold labeling

We report on application of flow cytometric and immunogold labeling techniques to purify and identify two types of murine epidermal dendritic cells: Langerhans cells (LC) and Thy-1-positive dendritic epidermal cells (Thy 1+-dEC). After density centrifugation of epidermal cell (EC) suspensions through Ficoll gradients. IA-positive LC and Thy 1+-dEC are labeled with monoclonal antibodies (fluorescein-conjugated anti-IAd for LC and anti-Thy 1.2-biotin, followed by avidin-phycoerythrin, for Thy 1+-dEC). The fluorescence-activated cell sorter (FACS) is then used to obtain 95-98% pure populations of these dendritic cells with a yield of 2-4 X 10(6) cells and a viability of 80-90%. A post-fixation, pre-embedding immunogold labeling technique using 15 nm and 40 nm colloidal gold particles is employed to identify LC and Thy 1+-dEC, respectively, to confirm the purity of the sorting and to estimate the number of IA antigenic sites per LC. With transmission electron microscopy, ultrastructural morphology of sorted LC is preserved; however, Birbeck granules are markedly diminished compared to the pre-sorted population of LC. In contrast, characteristic dense-core granules are readily visualized in sorted Thy 1+-dEC. Purification of epidermal dendritic cells by flow cytometry may be a useful technique to employ in functional studies of epidermal dendritic cells.