Retrograde tracing of neural pathways with a protein-gold complex. I. Light microscopic detection after silver intensification

In this study I have used a tracer complex made of wheat germ agglutinin horseradish peroxidase conjugate (WGA*HRP) coupled to colloidal gold for retrograde tracing of neuronal pathways at the light microscopic level. Visualization of the gold was achieved by silver precipitation (the gold silver intensification method) with gold particles acting as specific cores of nucleation. The presence of horseradish peroxidase in the protein conjugate allowed this method to be compared with classical histochemistry using tetramethylbenzidine as a chromogen. The gold silver intensification method proved to be reliable, specific and sensitive. It has been demonstrated to be useful with fixatives containing a high percentage of paraformaldehyde and compatible with histochemical procedures to show projections of transmitter specific pathways.     

A rapid and simple silver enhancement procedure for ultrastructural localization of the retrograde tracer WGAapoHRP-Au and its use in double-label studies with post-embedding immunocytochemistry

WGAapoHRP-Au is a colloidal gold conjugate of wheat germ agglutinin (WGA) coupled to enzymatically inactive (apo) horseradish peroxidase (HRP). This protein-gold complex has proven very useful for retrograde tracing studies in the nervous system (Basbaum and Menétrey: J Comp Neurol 261:306, 1987). To identify retrogradely labeled cells, the colloidal gold is made visible by silver intensification. As the tracer has no HRP enzymatic activity, it can be combined with HRP-based procedures (or with fluorescent methods) in a variety of multiple-label studies. Standard silver intensification procedures, however, are run at low pH and therefore are incompatible with good EM preservation; moreover, osmication of the tissue oxidizes the silver product, which is then lost in subsequent dehydration steps. This report describes a rapid and simple commercially available silver intensification procedure. The procedure is run at neutral pH and can be performed after osmication. The tracer is readily detected at the EM level and tissue preservation is excellent. This report also demonstrates how sections containing retrogradely labeled neurons can be stained with a post-embedding immunocytochemical method so that the transmitter content of synaptic inputs to these neurons can be identified.     

Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.     

Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry.

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.     

Metal-catalyzed oxidation renders silver intensification selective. Applications for the histochemistry of diaminobenzidine and neurofibrillary changes

Physical developers can increase the visibility of end products of certain histochemical reactions, such as oxidative polymerization of diaminobenzidine and selective binding of complex silver iodide ions to Alzheimer's neurofibrillary changes. Unfortunately, this intensification by silver coating is generally superimposed on a nonspecific staining originating from the argyrophil III reaction, which also takes place when tissue sections are treated with physical developers. The present study reveals that the argyrophil III reaction can be suppressed when tissue sections are treated with certain metal ions and hydrogen peroxide before they are transferred to the physical developer. The selective intensification of Alzheimer's neurofibrillary changes requires a pre-treatment with lanthanum nitrate (10 mM/liter) and 3% hydrogen peroxide for 1 hr. The diaminobenzidine reaction can be selectively intensified when physical development is preceded by consecutive treatments with copper sulfate (10 mM/liter, pH 5, 10 min) and hydrogen peroxide (3%, pH 7, 10 min). In peroxidase histochemistry, this high-grade intensification may help to increase specificity and reduce the threshold of detectability in tracing neurons with horseradish peroxidase or in immunohistochemistry when the peroxidase-antiperoxidase method is used.     

Protein-gold complexes as neuronal markers for long-term tracing studies

In this study, we have tested the possible use of protein-gold complexes as neuronal markers for long-term tracing studies in rat. The tracer we have used consisted of colloidal gold particles coupled to wheat-germ agglutinin apohorseradish peroxidase conjugate (WGA-apoHRP). The neuronal labeling was studied for survival periods of up to nineteen months following injection in the central nervous system. Maximal visualization of the gold particles was achieved through gold silver intensification. The tracer could be detected throughout the entire range of periods considered. The injection site consisted of a dense black core and retrogradely labeled cells were characterized by round black granules over the cell body. The retrogradely labeled cells were cytochemically characterized by demonstrating their transmitter content. Thus protein-gold complexes may be used as long-term neuronal markers compatible with the persistance of the vital functions of the labeled cells.     

Retrograde tracing of neural pathways with a protein gold complex. II. Electron microscopic demonstration of projections and collaterals

This paper describes a sensitive method for tracing neural connections at the electron microscopic (EM) level using a new compound produced through the coupling of colloidal gold particles to a wheat germ agglutinin horseradish peroxidase conjugate (the WGA*HRP-gold complex). Visualization of retrogradely labeled cells at the EM level was achieved either directly by gold particles scanning or after silver enhancement. By using different sizes of gold particles individually coupled to WGA*HRP and injected in different brain areas EM detection of multiple retrograde labeling was possible. Thus retrogradely labeled cells were first identified at the light microscopic level through HRP histochemistry with tetramethylbenzidine as a chromogen and then examined under the electron microscope after osmication and embedding. Gold particles were readily identified as electron dense, round dots in spherical grey vesicles. Identification of different sizes of gold particles often localized in the same vesicle established that the protein-gold complex can be used to study collateralisation of parental axons.     

Protein-gold complexes as neuronal markers for long-term tracing studies

Summary In this study, we have tested the possible use of protein-gold complexes as neuronal markers for long-term tracing studies in rat. The tracer we have used consisted of colloidal gold particles coupled to wheat-germ agglutinin apohorseradish peroxidase conjugate (WGA-apoHRP). The neuronal labeling was studied for survival periods of up to nineteen months following injection in the central nervous system. Maximal visualization of the gold particles was achieved through gold silver intensification. The tracer could be detected throughout the entire range of periods considered. The injection site consisted of a dense black core and retrogradely labeled cells were characterized by round black granules over the cell body. The retrogradely labeled cells were cytochemically characterized by demonstrating their transmitter content. Thus protein-gold complexes may be used as long-term neuronal markers compatible with the persistance of the vital functions of the labeled cells.     

Retrograde tracing of neural pathways with a protein gold complex

Summary This paper describes a sensitive method for tracing neural connections at the electron microscopic (EM) level using a new compound produced through the coupling of colloidal gold particles to a wheat germ agglutinin horseradish peroxidase conjugate (the WGA^*HRP-gold complex). Visualization of retrogradely labeled cells at the EM level was achieved either directly by gold particles scanning or after silver enhancement. By using different sizes of gold particles individually coupled to WGA^*HRP and injected in different brain areas EM detection of multiple retrograde labeling was possible. Thus retrogradely labeled cells were first identified at the light microscopic level through HRP histochemistry with tetramethylbenzidine as a chromogen and then examined under the electron microscope after osmication and embedding. Gold particles were readily identified as electron dense, round dots in spherical grey vesicles. Identification of different sizes of gold particles often localized in the same vesicle established that the protein-gold complex can be used to study collateralisation of parental axons.     

Rapid and reliable detection of 11 food-borne pathogens using thin-film biosensor chips

Traditional methods for identifying food-borne pathogens are time-consuming and laborious, so it is necessary to develop innovative methods for the rapid identification of food-borne pathogens. Here, we report the development of silicon-based optical thin-film biosensor chips for sensitive detection of 11 food-borne pathogens. Briefly, aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface, and then, biotinylated polymerase chain reaction (PCR) amplicons were hybridized with the probes. After washing and brief incubation with an antibiotin immunoglobulin G–horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate, biotinylated chains bound to the probes were visualized as a color change on the chip surface (gold to blue/purple). Highly sensitive and accurate examination of PCR fragment targets can be completed within 30 min. This assay is extremely robust, sensitive, specific, and economical and can be adapted to different throughputs. Thus, a rapid, sensitive, and reliable technique for detecting 11 food-borne pathogens was successfully developed.     

Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.     

Immune serum to protein molecular weight standards for calibrating Western blots

A method for calibrating proteins by Western blot analysis is described. Rabbit immune serum, specific for seven protein molecular weight standards, and goat anti-rabbit horseradish peroxidase conjugate are used to visualize standards after electrotransfer onto nitrocellulose. This method can be used as a reagent and procedure control.     

Versatility of anti-horseradish peroxidase antibody-gold complexes for cytochemistry and in situ hybridization: preparation and application of soluble complexes with streptavidin-peroxidase conjugates and biotinylated antibodies

Summary In previous studies we have employed a gold-labelled, affinity-purified polyclonal antibody against horseradish peroxidase (anti-HRP — gold) in the avidinbiotin peroxidase complex (ABC) technique and indirect labelled avidin-biotin methods. The gold-labelled antibody was used as final revealing reagent to replace the 3,3-diaminobenzidine (DAB) reaction by immunogold silver staining. The anti-HRP — gold reagent proved to be advantageous since blocking of endogenous peroxidase activity in the tissue sections was not further required and staining of superior contrast and resolution could be achieved in paraffin sections. In the present study we have optimized this technique by combining the last two incubation steps, i.e. HRP-conjugated streptavidin and anti-HRP — gold. Different ratios of the two reagents were tested empirically to establish the conditions for the formation of a soluble complex with optimal staining properties. Quantitative evaluation by densitometry of the staining intensity showed that the soluble streptavidin-HRP/anti-HRP — gold complex and the indirect labelled avidin-biotin method employing the gold-labelled anti-HRP antibody performed equally well. Thus, the availability of this complex simplifies the streptavidin-biotin immunogold technique for immunohistochemistry, lectin histochemistry and in situ hybridization and further demonstrates the versatility of anti-HRP — gold complexes.     

Performance characteristics of a reversible immunosensor with a heterobifunctional enzyme conjugate as signal generator

Factors that control the performance of a reversible immunosensor with an analyte (progesterone)-enzyme (horseradish peroxidase) conjugate as signal generator have been investigated. The conjugate is used in conjunction with two antibodies, which are specific to progesterone and to horseradish peroxidase, immobilized on two spatially separated polypropylene mesh discs. The conjugate and two antibodies are confined to an internal compartment of a microdialyzer by a semipermeable membrane. The small analyte from an external medium permeates across the membrane into the internal compartment where the analyte concentration determines the relative amounts of the bound conjugate on the two solid surfaces. By measuring two signals from the conjugate bound at two separate sites, we experimentally obtained time-response curves to a concentration pulse of the external analyte. A mathematical (kinetic) model describing the sensor system was developed and used for the determination of rate-limiting factors. In semicontinuous monitoring of the analyte concentrations, operation of the immunosensor with the enzyme conjugate as signal generator required special attention to (a) enzyme stability, (b) analyte permeation (dependence on medium components), and (c) kinetics related to the different accessibility to the same antibody of the small analyte (to be measured) vs. the larger counterpart on the enzyme conjugate (for signal generation). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 221-231, 1997.     

Alpha-bungarotoxin-horseradish peroxidase conjugate: preparation, properties and utilization for the histochemical detection of acetylcholine receptors.

A method is presented for the efficient conjugation of horseradish peroxidase to alpha-bungarotoxin. The 1:1 molar conjugate obtained is purified to completion by gel filtration on Sephadex G-100, followed by ion exchange chromatography on CM-Sephadex. The conjugate retains half of the activity of unmodified horseradish peroxidase and binds effectively to the nicotinic acetylcholine receptor of muscle. The conjugate is proven to be useful reagent for the histochemical staining of the receptor on muscle fibers for light and electron microscopy.     

Enzyme immunoassay for the measurement of 17alpha-estradiol 17-N-acetylglucosaminide in rabbit urine.

17alpha-estradiol 17-N-acetylglucosaminide (17alphaE2 17NAG) is an estrogen metabolite hitherto obtained only in rabbits. To gain insight into this unique conjugate, an enzyme immunoassay (EIA) was established by using antiserum elicited against 3-[3-(1-carboxypropyl)] ether of 17alphaE2 17NAG-bovine serum albumin conjugate; horseradish peroxidase, as a label; and 3,3',5,5'-tetramethylbenzidine, as a chromogen. The method proved to be specific, and the detection range of the assay was 0.20-10.00 ng/ml. A proposed double conjugate, 3-glucuronide of 17alphaE2 17NAG, was synthesized to validate the EIA. The EIA was applied to the determination of the urinary level of 17alphaE2 17NAG in male and female (pregnant and non-pregnant) rabbits with and without beta-glucuronidase-sulfatase preparation from Helix pomatia. The results showed that 17alphaE2 17NAG was mainly excreted as a double conjugate (17alphaE2 17NAG 3-glucuronide and/or 3-sulfate) and that its level varies during pregnancy.     

A comparison of silver ion to streptavidin coated microplates

Direct comparisons are made between covalently linked streptavidin and silver ion coated microplates. Both coatings can immobilize biotinylated molecules. Silver ion coated microplate wells can immobilize 1.8 times higher amounts of biotin labeled horseradish peroxidase. The quantitation range and capacity for the capture of horseradish peroxidase using biotin labeled horseradish peroxidase are also greater for silver ion coated microplates. Approximately twice as many anti-horseradish peroxidase antibodies can be immobilized per well using silver ion coated microplates. Higher capacities are presumed to be due to the smaller footprint of silver ions as compared to streptavidin. A direct comparison between the two coatings for a beta-galactosidase ELISA showed that while the silver ion coated microplates gave higher readings, the streptavidin coated microplates exhibited smaller well-to-well variation. However, higher well to well variation for the silver microplates is attributed to the high density of anti-beta-galactosidase antibodies on the microplates and the weak binding of clone GAL-13 to beta-galactosidase, rather than the silver coating. These studies suggest silver ion coated microplates are a desirable alternative to streptavidin plates for quantitative immunoassays.     

Formation of a bioconjugate composed of hemin, smectite, and quaternary ammonium chloride that is soluble and active in hydrophobic media

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) intercalated with a quaternary alkenylammonium compound, dioleyldimethylammonium chloride (DOA), to form a hemin-smectite-DOA conjugate. The hemin-smectite-DOA conjugate was soluble in organic solvents such as benzene and toluene to form a transparent colloidal solution with a light yellow color. Its absorption spectrum in benzene showed two bands, 600 and 568 nm, in the visible region and a sharp Soret band at 400 nm with the molar extinction coefficient of 7.5 x 10(4) M(-1) cm(-1). The formation of the conjugate of smectite and DOA was confirmed by X-ray diffraction analysis: the basal spacing, d(001), of hemin-smectite-DOA conjugate was 19 A which is an expansion of the interlayer space by 5 A based upon the basal spacing of smectite of 14 A. Hemin-smectite-DOA conjugate catalyzed the peroxidase-like reaction in organic solvents using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The temperature-dependent peroxidase-like activity of the conjugate was compared with peroxidase activity of horseradish peroxidase. The hemin-smectite-DOA conjugate exhibited higher activity as the temperature was increased from 30 to 70 degrees C, while horseradish peroxidase activity was reduced as the temperature was increased.     

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts

Carbohydrates have been suggested to account for some IgE cross- reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti- horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.      

Selective immunoprecipitate identification using monoclonal anti-rabbit IgG

A monoclonal mouse antibody directed against rabbit IgG has been conjugated with horseradish peroxidase and used to identify immunoprecipitates which contain rabbit antibodies. By combining a specific rabbit antisera with a general antiserum from another species (e.g., goat antiserum against human serum), immunoprecipitates containing the antigen(s) recognized by the rabbit antibodies have been selectively identified by colorimetric development of peroxidase activity. Since the monoclonal antibody is specific for rabbit IgG and nonprecipitating, the peroxidase conjugate can be included in the agarose with the primary antisera.