Applications of immunocolloids in light microscopy. III. Demonstration of antigenic and lectin-binding sites in semithin resin sections

Previous studies have demonstrated that antigens or lectin-binding sites can be localized in sections from paraffin-embedded tissues with protein A or lectins bound to colloidal gold or colloidal silver (Roth J: J Histochem Cytochem 30:691, 1982 and 31:547, 1983). In the present study the protein A-gold technique and lectin-gold complexes have been applied to semithin sections (0.5-1.5 micron) of Epon- or low temperature Lowicryl K4M-embedded rat pancreas, kidney and submandibular gland. The results show that an increase in resolution and, therefore, in amount of information can be obtained. The optimal mode of imaging was determined on sections without counterstaining. Bright-field illumination gives the maximum information about the staining signal, while phase-contrast and Nomarski differential interference contrast give predominantly structural and, to a lesser extent, staining information. Polarization epi- and transillumination microscopy is inferior in all aspects. The application of a battery of lectin-gold complexes to rat submandibular gland revealed a specific staining pattern for each lectin in acinar and excretory duct cells.     

Applications of immunocolloids in light microscopy. II. Demonstration of lectin-binding sites in paraffin sections by the use of lectin-gold or glycoprotein-gold complexes

A new procedure is presented for the light microscopic demonstration of specific sugar sequences of oligosaccharides in glycoconjugates by lectins combined with the colloidal gold marker system. Tissue sections from aldehyde-fixed and paraffin embedded rat kidney were stained either in a one-step method with lectin directly bound to particles of colloidal gold or in a two-step method using non-labeled lectin and glycoprotein labeled with colloidal gold. In both methods the presence of lectin-binding sites in the tissue sections is revealed by the appearance of a red coloration that is due to the accumulation of gold particles. The high specificity of the technique is combined with a good sensitivity and resolution as demonstrated by a differential plasma membrane staining in renal epithelial cells. The lectin-gold or glycoprotein-gold complexes remain stable for months and produce a permanent nonbleaching staining.     

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.     

"Golden blot"--detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complexes

Protein A bound to particles of colloidal gold is widely used for cytochemical localization of antigens. A method for detecting immune complexes on "Western blots" with protein A-gold has been developed. It involved the following steps: (i) electrophoretic separation of proteins, (ii) transfer to nitrocellulose, (iii) incubation with antibodies, and (iv) staining with protein A-gold. The amount of antigen detectable was 10-50 ng. The sensitivity could be improved by subsequent photochemical silver staining (1-10 ng detected), and was then comparable to autoradiograms using iodinated protein A.     

A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology.

In the present article we review several postembedding cytochemical techniques using the colloidal gold marker. Owing to the high atomic number of gold, the colloidal gold particles are electron dense. They are spherical in shape and can be prepared in sizes from 1 to 25 nm, which renders this marker among the best for electron microscopy. In addition, because it can be bound to several molecules, this marker has the advantage of being extremely versatile. Combined to immunoglobulins or immunoglobulin-binding proteins (protein A), it has been applied successfully in immunocytochemistry. Colloidal gold particles 5-15 nm in size are excellent for postembedding cytochemistry. Particles of smaller size, such as 1 nm, must be silver enhanced to be visualized by transmission electron microscopy. We have elected to review the superiority of indirect immunocytochemical approaches using IgG-gold or protein A-gold (protein G-gold and protein AG-gold). Lectins or enzymes can be tagged with colloidal gold particles, and the corresponding lectin-gold and enzyme-gold techniques have specific advantages and great potential. Using an indirect digoxigenin-tagged nucleotide and an antidigoxigenin probe, colloidal gold technology can also be used for in situ hybridization at the electron microscope level. Affinity characteristics lie behind all cytochemical techniques and several molecules displaying high affinity properties can also be beneficial for colloidal gold electron microscopy cytochemistry. All of these techniques can be combined in various ways to produce multiple labelings of several binding sites on the same tissue section. Colloidal gold is particulate and can easily be counted; thus the cytochemical signal can be evaluated quantitatively, introducing further advantages to the use of the colloidal gold marker. Finally, several combinations and multiple step procedures have been designed to amplify the final signal which renders the techniques more sensitive. The approaches reviewed here have been applied successfully in different fields of cell and molecular biology, cell pathology, plant biology and pathology, microbiology and virology. The potential of the approaches is emphasized in addition to different ways to assess specificity, sensitivity and accuracy of results.     

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.     

A Review of the Potential and Versatility of Colloidal Gold Cytochemical Labeling for Molecular Morphology

In the present article we review several postembedding cytochemical techniques using the colloidal gold marker. Owing to the high atomic number of gold, the colloidal gold particles are electron dense. They are spherical in shape and can be prepared in sizes from 1 to 25 nm, which renders this marker among the best for electron microscopy. In addition, because it can be bound to several molecules, this marker has the advantage of being extremely versatile. Combined to immunoglobulins or immunoglobulin-binding proteins (protein A), it has been applied successfully in immunocytochemistry. Colloidal gold particles 5–15 nm in size are excellent for postembedding cytochemistry. Particles of smaller size, such as 1 nm, must be silver enhanced to be visualized by transmission electron microscopy. We have elected to review the superiority of indirect immunocytochemical approaches using IgG-gold or protein A-gold (protein G-gold and protein AG-gold). Lectins or enzymes can be tagged with colloidal gold particles, and the corresponding lectin-gold and enzyme-gold techniques have specific advantages and great potential. Using an indirect digoxigenin-tagged nucleotide and an antidigoxigenin probe, colloidal gold technology can also be used for in situ hybridization at the electron microscope level. Affinity characteristics lie behind all cytochemical techniques and several molecules displaying high affinity properties can also be beneficial for colloidal gold electron microscopy cytochemistry. All of these techniques can be combined in various ways to produce multiple labelings of several binding sites on the same tissue section. Colloidal gold is particulate and can easily be counted; thus the cytochemical signal can be evaluated quantitatively, introducing further advantages to the use of the colloidal gold marker. Finally, several combinations and multiple step procedures have been designed to amplify the final signal which renders the techniques more sensitive. The approaches reviewed here have been applied successfully in different fields of cell and molecular biology, cell pathology, plant biology and pathology, microbiology and virology. The potential of the approaches is emphasized in addition to different ways to assess specificity, sensitivity and accuracy of results.     

Colored silver-intensified gold technique for light microscopy

The development of silver-intensified immunogold-labeled antibodies for light microscopy described by Fritz et al. (4) has been investigated. Principles and chemistries used in color photographic science have been applied to immunogold enhancement. In this technique, colloidal gold acts as the catalytic center for the reduction of silver ions to metallic silver with subsequent color development in the presence of hydroquinone. Silver ions and hydroquinone are adsorbed onto the surface of colloidal gold. The reduction of silver ions to metallic silver is further catalyzed by autometallography. The colored-SIG technique offers several advantages. It has sensitivity comparable to the silver-intensified gold (SIG) method and greater sensitivity than immunoenzymatic procedures, takes approximately one hour, results in one of three color reaction products (magenta, cyan, or yellow), and produces better contrast between the reaction products and the background (Figure 1). Thus, this method should prove useful in double- and even triple-staining procedures.     

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.     

Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.     

A double protein A-gold-silver staining method for tissue antigens in light microscopy

Summary A double staining method has been developed for light microscopic immunohistochemistry. The technique consists of a sequence of protein A-gold-silver procedures, in which the reaction products are visualized in black and brown colours. The results obtained in the rat pancreatic islets and adenohyophysis, in combination with a variety of histochemical controls, substantiate both the specificity and reliability of this method. The double staining method is simple and economical, since the two differently coloured reaction products are obtained by a common mechanism of colouration (physical development) using a single probe, protein A-gold.     

Epipolarization Microscopic Immunogold Assay: a Combination of Immunogold Silver Staining, Enzyme-Linked-Immunosorbent Assay and Epipolarization Microscopy

We describe a new immunoassay which combines an immunosorbent assay, Immunogold silver staining and epipolarization microscopy. Our new assay procedure features multiple samples on a single microscope slide, and high sensitivity of epipolarization microscope for detection of silver-enhanced colloidal gold as a final immunoassay product. We call the new immunoassay “on slide immunogold assay” (OSIGA). This new method uses biotinylated antibody and streptavidin-gold reaction with silver enhancement technique. With OSIGA it is possible to investigate 30 samples on a single microscopic slide. Our preliminary studies used 10-20 μ1 samples and detected nanogram quantities of a standardized protein solution. Unlike enzyme linked immunosorbent assay (ELISA), which has a limited time for reading the final color products, the OSIGA specimens can be dried or resin mounted for longer storage and future reference.     

Lectin--digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry.

We report the use of a novel hapten system for lectin cytochemistry. Various lectins conjugated to the steroid hapten digoxigenin (DIG) and monospecific anti-digoxigenin antibodies were applied for the light and electron microscopic detection of glycoconjugates in tissue sections. Both IgG and Fab' anti-DIG antibodies were complexed to particles of colloidal gold and compared to commercially available alkaline phosphatase and horseradish peroxidase conjugated Fab' as general second step reagents. The three different markers performed equally well on paraffin sections whereas the gold-labeled antibodies were superior reagents for semithin and ultrathin sections of Lowicryl K4M embedded tissues. In conjunction with the latter marker, no pretreatment to abolish endogenous enzyme activity was necessary. At the light microscope level, gold signal amplification by the photochemical silver reaction was required. DIG, in contrast to biotin, does not occur in animal tissues thus eliminating the need for blocking reactions prior to lectin incubation. Compared to affinity techniques using glycoprotein-gold complexes as second step reagent the DIG hapten system required smaller amounts of lectins. The staining patterns were indistinguishable from those obtained in other lectin-gold techniques and the specificity of the labeling could be demonstrated in sugar inhibition tests.     

Simplified purification and testing of colloidal gold probes

A novel efficient method for purifying and testing colloidal gold probes has been developed. The method consists of concentrating colloidal gold particles conjugated to IgG or protein A in dialysis bags over silica gel and purifying them by gel chromatography on small columns of Sephacryl S-400. Fractions collected are tested by paper immunocytochemical models. Comparisons to gold probes purified by conventional ultracentrifugation documents that ultrastructural staining intensities and total yield of gold probes is the same, but that the chromatographically purified gold probes are less prone to aggregation or clumping. The method has been extensively used for preparing conjugates of 5, 10 or 15 nm gold particles with antirabbit immunoglobulins but has also been exploited for preparing streptavidin-gold conjugates, protein A-gold conjugates and antirabbit immunoglobulin-silver conjugates.     

Investigation of immunogold-silver staining by electron microscopy

Deposition of metallic silver on colloidal gold immunoreagents has been shown to be a very sensitive immunostaining technique capable of detecting low levels of immunoreactivity in tissue sections. Using electron microscopy we have shown that immunolabelling is highest with small sizes of gold which can penetrate sections better and achieve higher densities of particles in the section than larger particles. Chemical permeabilisation of the embedding medium aids the penetration of colloidal gold. The silver enhancement step in immunogold-silver staining was shown to be progressive, allowing optimisation of staining and the selection of the final size of silver deposits required. Some poorly understood features of the technique are rationalised and the additional knowledge gained will aid the wider application of this method.     

Silver enhancement of protein A-gold probes on resin-embedded ultrathin sections

Summary 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 magmfication, with a negligeable background staining.The present technique can be useful whenever it is necessary to use the smallest gold probes today available.Supported by contract No. 85.02038.44 from the National Research Council, Rome, Progetto Finalizzato Oncologia     

Immunogold silver staining for light microscopy

 The immunogold silver staining method (IGSS) is widely used as a sensitive and specific immunohistochemical visualisation technique. IGSS involves the specific deposition of metallic silver at the site of immunogold labelling and provides a means of visualisation at low magnification by light or electron microscopy. Silver developers for IGSS rapidly deposit metallic silver only at the site of heavy metals, including gold and silver, because of their catalytic activity. The developing solution contains the silver ions and reducing agent necessary for this reaction. Using different silver salts as ion donors and by selecting an appropriate temperature and pH, visible amounts of silver can be deposited in a few minutes at the site of colloidal gold labelling while little non-specific background deposition occurs. Inclusion of protective colloids in the solution can also be used to control the reaction. Although studies of the chemical basis of silver deposition around unlabelled colloidal gold date back to 1939, immunogold enhancement by silver was established in 1983. The IGSS method evolved from the combination of disparate photographic, histochemical and immunogold techniques which have been effectively combined and optimised over the last 10 years to provide a visualisation system which is well suited to many immunohistochemical studies. Accepted: 29 April 1996     

Detection of the Neu5 Ac (alpha 2,3) Gal (beta 1,4) GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid in alpha 2,3- and alpha 2,6-linkage

The Maackia amurensis leukoagglutinin has been shown to react specifically with the Neu5Ac (alpha 2,3) Gal sequence of asparagine-linked complex type oligosaccharides. We report here the preparation of Maackia amurensis lectin-gold complexes and their application for light and electron microscopic detection of the Neu5 Ac (alpha 2,3) Gal sequence in various tissues. The use of the lectin directly gold labeled was superior to a two-step cytochemical affinity technique using a fetuin-gold complex. The Maackia amurensis lectin-gold staining was inhibited by pre-incubation of the lectin-gold complexes with 50 mM alpha 2,3 sialyllactose, whereas alpha 2,6 sialyllactose up to concentrations of 1 M had no effect, thus demonstrating the high specificity of the histochemical staining. In addition to N-glycanase-sensitive asparagine-linked oligosaccharides, beta-elimination-sensitive serine/threonine-linked oligosaccharides could be detected. Data are presented which show that cellular staining patterns obtained with Maackia amurensis lectin-gold complexes may differ from those with elderberry bark lectin-gold, which detects the Neu5 Ac (alpha 2,6) Gal/GalN Ac sequence. Electron microscopic double labeling for direct study of the differential distribution of the Neu5 Ac (alpha 2,3) Gal and Neu5 Ac (alpha 2,6) Gal sequences is reported. Therefore, the availability of two sialic acid binding lectins with different linkage specificity for histochemistry provides the first opportunity to study tissue and cell type expression of these terminal sequences of glycoproteins.     

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.     

Immunogold silver staining method for light microscopic detection of peripheral blood lymphocyte surface antigens with monoclonal antibodies.

Labelling of peripheral blood Lymphocytes surface antigens was carried out using the method of colloidal gold, enhanced with silver staining. Instead of PBS the minimal essential medium (MEM) according the Eagle, pH 7.2, was used rinsing of isolated lymphocytes. Visibility of positive reactions on lymphocytes at application of both mentioned media was the same. Positive reaction at demonstration of p24 BLV on cells acquired the from of black cap while the IgG expression was observed in the from of diffuse dispersion of colloidal gold on cells. Differences between the application of individual media were observed in the shape of peripheral lymphocytes in smears. Utilization of Eagle's MEM resulted in more uniform shapes and optically smooth surfaces when viewed under a light microscope.