Labelling of colloidal gold with protein

Summary Markers prepared by labelling colloidal gold with macromolecules such as lectins, antibodies and protein A are gaining wise acceptance both in transmission and scanning electron microscopy. However detailed information on the process and extent of adsorption of macromolecules onto gold particles are still lacking. The adsorption isotherm of protein onto gold particles was studied quantitatively using goat -lactoglobulin (L) tritiated in vivo. When this protein was modified chemically by iodination with ¹²⁵I, the adsorption isotherm was not significantly different (Langmuir type for monolayer). In the presence of saturating amount of L, a maximum of 13–14 molecules was adsorbed per particle of 12 nm in diameter for a theoretical maximum of 20 (compact monolayer). Ellipsometric measurements on nickel-coated slides indicated that L was adsorbed onto metallic surfaces as a compact monolayer. The molecules were irreversibly adsorbed on gold particles, kept to a large extent their capacity to bind anti-L antibodies and could not be displaced by polyethylene glycol, a stabilizer commonly used in the preparation of gold markers. Only markers labelled with more than 5 L molecules per particle could be completely bound by immobilized anti-L antibodies. Preliminary data indicated that the energetics of adsorption of L onto colloidal gold was in agreement with that expected from the mutual interaction of surface and adsorbate.     

Suitability of Different Protein A-Gold Markers for Immunogold-Silver Staining in Paraffin Sections

An incubation protocol to immunolabel Lowicryl semithin sections was applied to paraffin probes. To improve the labeling density, colloidal gold complexes of different preparations and sizes were compared. The type of colloidal gold preparation used was found to affect the specificity of the immunostaining. Gold colloid of 5 nm diameter particle size prepared with white phosphorus minimized nonspecific background labeling of β-casein in paraffin embedded sections of the mammary epithelium of pregnant mice. Gold colloids of 5 nm and 9 nm diameter particle size prepared in varying concentrations of tannic acid generated significant nonspecific staining in similar tissue preparations.     

The preparation of protein A-gold complexes with 3 nm and 15 nm gold particles and their use in labelling multiple antigens on ultra-thin sections

Summary The preparation of a protein A-gold complex (pAg₃) using 3 nm gold particles and its application for labelling of intracellular antigens on thin sections is reported. The 3 nm gold particle is the smallest metal particle currently available for cytochemistry and permits a higher resolution of the pAg technique. Furthermore, it can be used in double labelling experiments in conjunction with a pAg complex prepared from 15 nm gold particles. For double labelling, the pAg₃ complex must be used for staining of the first antigen since otherwise a non-specific co-labelling of the two pAg complexes results.     

Suitability of Different Protein A-Gold Markers for Immunogold-Silver Staining in Paraffin Sections

An incubation protocol to immunolabel Lowicryl semithin sections was applied to paraffin probes. To improve the labeling density, colloidal gold complexes of different preparations and sizes were compared. The type of colloidal gold preparation used was found to affect the specificity of the immunostaining. Gold colloid of 5 nm diameter particle size prepared with white phosphorus minimized nonspecific background labeling of β-casein in paraffin embedded sections of the mammary epithelium of pregnant mice. Gold colloids of 5 nm and 9 nm diameter particle size prepared in varying concentrations of tannic acid generated significant nonspecific staining in similar tissue preparations.     

Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning

Summary Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at 0° C, such as progressive lowering of temperature (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.Abbreviations AB antibodies - AED aminoethyldextran - Au₁₀ colloidal gold (10 nm diameter) - BSA bovine serum albumin - EtOH ethanol - FA formaldehyde - GA glutaraldehyde - LN₂ liquid nitrogen - MeOH methanol - pA protein A - Pipes piperazine-N,N-bis(2-ethanesulphonic acid) - PLT progressive lowering of temperature - PVP polyvinylpyrrolidone - UA uranylacetate     

Labelling of colloidal gold with IgE

Summary Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal ¹²⁵I-IgE antibovine milk -lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1–8.8 (28 nm in particle diameter). At low coverage of the particles (5 molecules per particle), the isotherm was independant of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2–5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mappings of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4°C. Surface clusters developed when the cells were incubated at 37°C.     

Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry

Summary Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG—gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase—isomaltase. The labelling efficiency of RPGG was compared to that of protein A—gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12nm particles, 10.3 and 6.2 particles/µm of length of microvillar membrane, 3.5 and 1.0 particles/µm² of Golgi profile and 5.9 and 2.0 particles/µm² of multivesicular body profile; and for the 6nm particles, 49.6 and 15.7 particles/µm of length of microvillar membrane, 24.4 and 5.0 particles/µm² of Golgi profile and 25.4 and 3.4. particles/µm² of multivesicular body profile. Controls showed very little non-specific gold labelling (<0.02 gold particles/µm² of section).     

Analysis of colloidal gold probes by isoelectric focusing in agarose gels

Colloidal gold particles of different size (3-20 nm in diameter) were prepared by tannic acid-citrate and citrate reduction methods. From these colloids, different probes were prepared using sheep anti-rabbit antiserum, sheep anti-rabbit IgG, bovine serum albumin, polyethylene glycol, and protein A as the primary stabilizers and polyethylene glycol and/or bovine serum albumin as secondary and tertiary stabilizers, in different combinations. The probes were analyzed by isoelectric focusing in agarose gels, which allow the migration of particles in the size range 3-20 nm. (P. Sewer and S. J. Hayes, 1986, Anal. Biochem. 158, 72-78). Isoelectric focusing revealed that the surface charge of colloidal gold probes is dependent upon the size of the gold particle, the reduction method used, the primary ligand, and the pH at which this is adsorbed, as well as upon the secondary and tertiary stabilizers used. It is proposed that such differences in surface charge may underlie the different results which may sometimes be observed in colloidal gold labeling, especially when novel ligands are used.     

Fine localization of serine: pyruvate aminotransferase in rat hepatocytes revealed by a post-embedding immunocytochemical technique

Summary Immunocytochemical localization of serine: pyruvate aminotransferase (SPT) in rat hepatocytes was studied using a protien A-gold technique. Rat liver was fixed by perfusion. Vibratome sections (100 m thick) of the liver were embedded in Epon or Lowicryl K4M. Ultrathin sections were incubated with antiSPT, followed by protein A-gold complex. Gold particles representing the antigenic sites for SPT were seen in three subcellular compartments, peroxisomes, mitochondria, and cytoplasm. In the control experiments the specificity of the immunolabelling was confirmed. Quantitative analysis of the labelling density showed that main subcellular compartments containing SPT are mitochondria and peroxisomes. In addition, the gold particles distributing in the cytoplasm were 16%–29% of the total labelling. The result indicated that the cytoplasm also contains SPT with a low density.     

Silver-enhanced colloidal gold probes as markers for scanning electron microscopy

Summary Silver enlargement of small colloidal gold particles has been extensively used for the light microscopical visualization of gold probes. Very recently, a few investigators have employed physical developers in electron microscopy (both pre-embedding and on-grid staining methods). We now demonstrate that physical development of small colloidal gold particles advantageously can be exploited for labelling biological surfaces in scanning electron microscopy. This novel application of silver enhancement of colloidal gold particles is characterized by a high detection efficiency. Thus, specimens are labelled with small gold probes affording high immunocytochemical efficiency but being impossible to detect with the present scanning microscopes. These particles are subsequently scanning electronmicro-scopically visualized by silver enhancement.Presented in part at the International Symposium on Biological Regulation of Cell Proliferation, 9th International Chalone Conference, Milano, Italy, March 3–6, 1986     

Chitosan-colloidal gold complexes as polycationic probes for the detection of anionic sites by transmission and scanning electron microscopy

Summary A new cationic colloidal gold complex has been developed for ultrastructural localization of cell surface anionic sites by transmission and scanning electron microscopy. The marker is prepared by labelling gold particles of suitable sizes (6 to 70 nm in diameter) with chitosan, a polymer of (14)-linked d-glucosamine. Using human red blood cells as a model, chitosan-gold complexes were shown to be specific for anionic sites and at pH 2 for sialic acid residues. The binding capacity of complexes of different sizes with carboxymethyl and phosphorylated celluloses was examined as a function of pH and ionic strength. The results indicated that these complexes can be used under acidic conditions as well as in physiological buffers. The complexes were further tested by transmission and scanning electron microscopy in detecting anionic sites on cells of various origins such as Escherichia coli, Lactobacillus maltaromicus, Lactobacillus reuteri, Saccharomyces cerevisiae, Saccharomyces rouxii, Schizosaccharomyces pombe, Fusarium oxysporum, Catharantus roseus.     

Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning. Analysis of the secretory organelle matrix of Paramecium trichocysts.

Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at greater than or equal to 0 degree C, such as "progressive lowering of temperature" (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.     

Effect of particle size on labeling density for catalase in protein A-gold immunocytochemistry

Effect of particle size on labeling intensity in protein A-gold immunocytochemistry was studied. Catalase labeling of rat liver peroxisomes was used as a labeling model. Ultra-thin sections of Lowicryl K4M-embedded rat liver were stained for catalase with protein A-gold (pAg) probes. Five different sizes of colloidal gold probes, from 5 nm to 38 nm in diameter, were prepared. Labeling intensity decreased as the particle size of the pAg probes increased. The highest labeling was obtained by the 5-nm pAg probe and the lowest by the 38-nm pAg probe. Quantitative analysis also showed that labeling density was inversely proportional to the size of gold particles. The results suggest that the pAg probe with small gold particles has high sensitivity.     

Biological synthesis of gold nanoparticles by the xylotrophic basidiomycete Lentinula edodes

This is the first study to demonstrate that the medicinal basidiomycete Lentinula edodes can reduce gold (III) ions from hydrogen tetrachloaurate (chloroauric acid) H[AuCl₄] to the elementary state with the formation of spherical nanoparticles (nanospheres). When a culture was grown under submerged conditions in the presence of chloroauric acid, the appearance of an intense purple-red color of L. edodes filamentous hyphae was recorded, which indicates that gold ions were reduced to gold nanoparticles. Using transmission electron microscopy and X-ray fluorescence, we observed accumulation of colloidal gold by the fungal mycelium in the form of electron-dense nanospheres of 5 to 50 nm in diameter on the surface and inside fungal cells.     

Labelling of colloidal gold with IgE. A quantitative study using monoclonal IgE anti-beta-lactoglobulin and evaluation of the biological activity of the gold complex with RBL-1 cells

Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal 125I-IgE antibovine milk beta-lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1-8.8 (28 nm in particle diameter). At low coverage of the particles (less that or equal to 5 molecules per particle), the isotherm was independent of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2-5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mapping of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4 degrees C. Surface clusters developed when the cells were incubated at 37 degrees C.     

Effective diameters of protein A-gold and goat anti-rabbit-gold conjugates visualized by field emission scanning electron microscopy.

High-voltage (15-30 kV) field emission scanning electron microscopy (FESEM) was used to evaluate the effects of gold particle size and protein concentration on the formation of protein-gold complexes. Six colloidal gold sols were prepared, ranging in diameter from 7.6 to 39.8 nm. The minimal protecting amounts (m.p.a.) of protein A and goat anti-rabbit antibody (GAR) were experimentally determined. Gold particles were conjugated at the m.p.a., one half the m.p.a., and ten times the m.p.a. for both proteins, and protein-gold complexes prepared for FESEM. The smallest colloidal gold particles required the most protein per milliliter of gold suspension for stabilization. Transmission electron microscopy was found to be the preferred method for accurate sizing of gold particles, whereas FESEM of protein-gold complexes permitted visualization of a protein halo around a spherical gold core. Protein halo width varied significantly with changes in gold particle size. Measurements of protein halos indicated that conjugation with the m.p.a. of protein A resulted in the thickest protein layers for all gold sizes. GAR conjugation with the m.p.a. again produced the thickest protein layers. However, GAR halos were significantly smaller than those obtained with protein A conjugation. The proteins used showed similar adsorption patterns for the larger gold particles. For smaller gold particles, proteins may act differently, and these complexes should be further characterized by low-voltage FESEM.     

Light microscopic localization of silver-enhanced liposome-entrapped colloidal gold in mouse tissues

Silver-enhanced liposome-entrapped colloidal gold was developed for light microscopic localization of liposomes. Preparation of colloidal gold entrapped in liposomes was achieved by a modified method of Hong, et al. (1983) Biochim. Biophys. Acta 732, 320-323). In this report, a gold chloride/citrate solution of low pH (3.4) was used to inhibit the formation of gold granules during the liposome preparation. The diameter of most liposomes ranged from 80 to 100 nm. Following liposome preparation, the pH was adjusted to 6, and the temperature increased to 55 degrees C. The majority of the liposomes contained one to three gold particles. Liposomes were injected into mice via tail vein; 24 h later, tissues were collected. Sections were processed for silver enhancement of the gold particles and examined by light microscopy. Silver-enhanced gold particles were clearly observed in both liver and implanted tumor. Localization was confirmed by electron and fluorescence microscopy. Thus, we have shown that silver enhancement of colloidal gold liposomes is a direct and sensitive method for tracing the fate of liposomes in vivo, providing minimal background interference and a good definition of various cell types.     

Quantitative immunohistochemical evidence of a functional gradient of chondroitin 4-sulphate/dermatan sulphate, developmentally regulated in the predentine of rat incisor

A quantitative examination was carried out on the early and mature stages of dentinogenesis in the rat incisor, using a post-embedding immunogold labelling with an anti-chondroitin 4 sulphate/dermatan sulphate antibody (2B6). At a very early stage of predentine formation, before polarizing odontoblasts have established junctional complexes, immunolabelling was weak. In contrast, when polarized odontoblasts established distal junctional complexes, immunolabelling in predentine was uniform and threefold denser than in initial predentine. The same gold particle density was found in the non-mineralized mantle dentine. During circumpulpal dentine formation, a gradient was seen in predentine, a larger number of gold particles being scored in the proximal zone compared with the distal region adjacent to the mineralization front. In circumpulpal dentine, some labelling was found within the lumen of the tubules and in the bordering dentine around the tubules. A few particles were also detected in intertubular matrix after demineralization. Together, these data provide evidence for a developmentally regulated gradient during the transition between mantle and circumpulpal dentine, and also in a more mature part of the tooth, a functional gradient that probably plays a role in the process of mineralization. © 1998 Chapman & Hall     

Microwave-stimulated incubation in immunoelectron microscopy: A quantitative study

Summary Microwave irradiation has been applied to reduce the immunogold staining time of ultrathin sections of Lowicryl embedded specimens. Labelling has been stimulated by microwave irradiation during incubation with 10nm colloidal gold particls coated with either goat anti-mouse antibodies (GaM-gold) or goat anti-rabbit antibodies (GaR-gold) and has been compared with control incubations. Quantification has been performed on cytoplasmic membranes or lysosomes labelled with a primary antibody. Counting the gold particles over specific and non-specific sites in electron micrographs and electron microscopic images by IBAS 2000 revealed that irradiation of 25 l droplets both at 80W and 150 W resulted in an accelerated immunogold labelling, while the non-specific background levels were not increased. A plateau level in immunogold labelling intensity was reached after 25 min incubation under microwave irradiation at 150W as compared to 120 min incubation without microwaves. No improvement in localization sharpness of immunogold labelling on membranes was achieved by microwave irradiation. The microwave-mediated acceleration of immunogold staining may be considered as an example of a staining method with a restricted thermal action on microvolumes as indicated by direct temperature measurements using a fibre-optic thermometer.     

Enzyme-gold cytochemistry of seed xyloglucans using two xyloglucan-specific hydrolases. Importance of prior heat-deactivation of the enzymes

Summary Two pure, homogeneous xyloglucan-hydrolyzing enzymes from germinated nasturtium seeds have been used to localize xyloglucans specifically in seed cell walls. The enzymes, a novelendo (14)--d-glucanase which shows absolute specificity towards xyloglucans and a -d-galactosidase which is capable of removing galactosyl residues from polymeric xyloglucans, were used to stabilize gold sols. The complexes were applied to ultrathin sections of nasturtium (Tropaeolum majus L) and tamarind (Tamarindus indica L) seeds. The gold complexes prepared from the active enzyme proteins retained enzyme activity, and such complexes gave extremely weak section-labelling or no labelling at all. When the enzymes were subjected to heat-deactivation before being used to stabilize the gold sols, gold complexes were obtained which lacked enzyme activity, but which gave strong, specific labelling of xyloglucans in ultrathin sections. The specificity of the labelling was checked by substrate-competition, by pretreatment of sections with the active and heat-denaturated enzymes and by comparing the labelling of xyloglucan-containing storage cells with other cell types in the same section. The labelling was maximal at the pH which was optimal for the active enzyme. We conclude that the enzyme-gold complexes which retain high activity against the substrate to be localized are likely to be unsuitable as cytochemical probes because they may causein situ substrate modification. In the case of the enzyme complexes described here the specific localization obtained with the gold complexes prepared from heat deactivated enzymes may be attributable to the retention by the heat-treated enzymatically-inactive proteins of substrate recognition. Alternatively, some recovery of the native configuration of the heat-denatured protein may have occurred on adsorption to the surface of the colloidal gold particle.