Effect of particle size on labeling density for catalase in protein A-gold immunocytochemistry (original) (raw)
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The Histochemical Journal, 1989
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//~m of length of microvillar membrane, 3.5 and 1.0 particles/[xm 2 of Golgi profile and 5.9 and 2.0 particles//~m 2 of multivesicular body profile; and for the 6 nm particles, 49.6 and 15.7 particles//~m of length of microvillar membrane, 24.4 and 5.0 particles//~m 2 of Golgi profile and 25.4 and 3.4. particles//~m 2 of multivesicular body profile. Controls showed very little non-specific gold labelling (<0.02 gold particles//~m 2 of section).
Journal of Histochemistry & Cytochemistry, 1999
The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific -oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.
Protein A Gold-Silver Staining Method for Light Microscopic Immunohistochemistry
Archives of Histology and Cytology, 1985
A new sensitive method has been established for light microscopic immunohistochemistry. It involves a protein A gold technique instead of immunoglobulin-gold method, followed by an improved procedure of physical development. In this procedure, two selected reagents were employed: bromohydroquinone, a more potent developing agent than hydroquinone, and specifically purified gum arabic solution. The results obtained by this method in the pancreatic islet tissues of the rat under a variety of histochemical control conditions have substantiated both the high specificity and fidelity of this method.
Journal of Immunological Methods, 2003
Rapid and sensitive screening for confirming the reactivity of reagents, before proceeding for electron microscopy, is highly desirable. ELISA-based methods have been shown to be highly efficient and successful for rapid prescreening and optimization of immunological as well as sample-processing reagents for the sensitive detection and quantitation of antigen by electron microscopy. The drawback of these methods lies in their inability to provide any information regarding the gold conjugate used for the final observed and measured signal. In this work, we demonstrate a simple and rapid, solid-phase method in ELISA format that is also suitable for evaluation and optimization of the gold conjugate. We have demonstrated the utility of this technique by screening for Vitreoscilla hemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold-labeling transmission electron microscopy (TEM) of cell sections. The sensitivity of detection and quantitation of antigens by immuno-electron microscopy depends upon the assay procedure being optimized to obtain the best possible signal. Our study indicates that evaluation of gold conjugate by the solid-phase assay could help in the rapid optimization of this reagent for immunogold localization and quantification of antigens by TEM. D
Microchimica Acta, 2011
We have developed a column gel-based immunoassays for the model analyte benzo[a]pyrene (B[a]P), and have evaluated three different kinds of labels, i.e., horseradish peroxidase, (HRP), colloidal gold (AuNPs), and quantum dots (QDs) with respect to rapid visual on-site testing. In case of HRP, a derivative of B[a]P-BA (benzo[a] pyrenebutyric acid) was directly coupled to HRP. In case of QDs and AuNPs, protein conjugates of B[a]P-BA were synthesized and used for surface functionalization. With the latter, previous coverage of the gold surface with 11mercaptoundecanoic acid turned out to be advantageous in terms of antibody recognition and of preventing nonspecific binding to the gel layer. The assays based on the use of particle labels requires 4 consecutive working steps only, while those based on HRP require 5 additions of reagent. The lower limit of detection for B[a]P is 5 ng L −1 in case of using HPR or QDs as a label, but 25 ng L −1 when using AuNPs. We believe that the use of QDs constitutes the most promising label for qualitative and semi-quantitative gelbased immunoassays.
Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry
Journal of Histochemistry & Cytochemistry, 1992
In pre-embedding EM immunocytochemistry with gold probes, the gold must be small enough to penetrate through cell membranes treated with mild detergents. Antibodies labeled with small gold probes (1-1.4 nm) are too small to be resolved in thin sections but can be seen if they are silver-enhanced after the gold has bound to the antigens in the cells. We investigated several aspects of gum arabic-silver lactate-hydroquinone enhancement solution (Danscher solution) by examining gold-conjugated antibodies embedded in agar, sectioned on a vibrotome, and enhanced with different solutions. The rate of silver enhancement was optimized in 50% gum arabic and 200 mM HEPES buffer, pH 5.8. We also examined chemicals used as developers and found that N-propyl gallate (NPG) gave a more uniform development than the routinely used hydroquinone (HQ). The diameter of the silver-enhanced particles after incubation in osmium tetratoxide (OSO4) decreased somewhat with longer incubation time and higher pe...