Gold-labeled Goat Anti-rabbit IgG Research Articles

Immunocytochemical localization of MG1 mucin in human bulbourethral glands

Salivary mucins MG1 and MG2 have been found in the oral cavity where they perform several functions such as the formation of the mucous layer covering the oral mucosa and teeth. Recent studies have demonstrated their presence in other organs and tissues. The aim of this study was to determine their expression in human bulbourethral (Cowper's) glands. Normal bulbourethral glands were obtained at surgery and fixed in a mixture of 1% paraformaldehyde-1.25% glutaraldehyde in 0.1 M cacodylate buffer and embedded in Epon resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells were intensely reactive with anti-MG1, whereas no labeling was detected for MG2. These results indicate that MG1 is not exclusively a salivary component and furthermore show that bulbourethral glands represent a significant source of the MG1 detected in human seminal plasma.

Immunolocalization of myocilin protein in the anterior eye of normal and primary open‐angle glaucomatous dogs

The presence of myocilin was investigated in a colony of Beagles, a canine model for inherited primary open-angle glaucoma (POAG). The myocilin protein was localized in the normal and glaucomatous canine eyes by immunohistochemistry and immunocytochemistry. Paraffin- and plastic-embedded specimens from the anterior uveas of 10 Beagles with inherited glaucoma (3 months to 13 years old) and 6 age-matched normal dogs were sectioned, and were then incubated with primary antibody, rabbit polyclonal antihuman MYOC IgG, overnight at 4 degrees C. Specimens were incubated with secondary antibody with one of the following: biotinylated link followed by peroxidase-labeled streptavidin and then by substrate-chromogen for light microscopy; fluorescent marker Texas red; or 18 nm colloidal gold-labeled goat antirabbit IgG for transmission electron microscopy. With normal, pre- and early glaucomatous canine specimens, cell membranes of smooth muscle cells of the iris and ciliary body stained positively, as well as most resident stromal and vascular endothelial cells. The cytoplasm of cells within the nonpigmented ciliary epithelium of the ciliary body processes stained intensely, being weaker along the pars plana. Trabecular meshwork (TM) cells and surrounding extracellular matrix labeled, as well as the sclera adjacent to the angular aqueous plexus. In specimens with moderate and advanced glaucoma, greater intensity of staining was observed within TM cells and adjacent sclera, and portions of the nonpigmented epithelium of the ciliary processes. Fibrinous material labeled intensely within the posterior chamber. Myocilin in the normal and glaucomatous canine eye was successfully immunolocalized. These findings with regard to the normal eye are nearly identical to those previously reported in humans, and support the original hypothesis that there is an increase in both accumulation and localization of myocilin in glaucomatous canine eyes. It also supports the possibility that changes in the activity of myocilin within the aqueous humor outflow pathway of individuals with spontaneous glaucoma are associated with the rise of intraocular pressure and subsequent development of this disease, but may not be the primary event in the initial raise in intraocular pressure in POAG in the Beagle.

Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy.

We performed indirect immunogold electron microscopy (EM) for immunological identification and characterization of hepatitis C virus (HCV). To clarify the morphology of HCV, an indirect immunogold EM of two plasma samples from patients with high HCV RNA titers was carried out using antibodies specific for the putative HCV envelope protein (E) 1. Spherical virus particles 55-65 nm in diameter with delicate spike projections were detected in the 1.14-1.16 g/ml fractions after sucrose density gradient centrifugation. Polyclonal and monoclonal antibodies to the putative HCV E1 specifically recognized these particles. In addition, immunogold EM of the samples was also performed to uncover the morphology of HCV core particles. Spherical particles 33-40 nm in diameter (average, 37 nm) were detected in the 1.22- to 1.25-g/ml fractions by conventional EM after sucrose density gradient centrifugation. Immunogold EM using rabbit polyclonal antibody (RR8) specific for the putative HCV core protein and colloidal gold-labeled goat antirabbit IgG showed binding of the gold particles with RR8. Some of the HCV core particles showed icosahedric morphology. Optical rotation technique showed that the HCV core particles exhibit sixfold symmetry and that the length of the regular hexagon side is approximately 20 nm, suggesting that they have an icosahedric structure. Further, the detection limit of the indirect immunogold EM was evaluated in 11 plasma samples from chronic hepatitis B patients with different degrees of hepatitis B virus (HBV) DNA titers using antihepatitis B surface antigen antibody. The study showed that the detection limit of virus using this method is 10(7) virions/ml.

Silver-enhanced colloidal gold metalloimmunoassay for Schistosoma japonicum antibody detection

A silver-enhanced colloidal gold metalloimmunoassay has been proposed for the determination of Schistosoma japonicum antibody (SjAb) in rabbit serum. The adult worm antigen of S. japonicum (SjAg) was adsorbed passively on the walls of a polystyrene microwell and then reacted with the desired SjAb. The colloidal gold-labeled goat anti-rabbit IgG secondary antibody was adsorbed on the walls of the polystyrene microwells through the reaction with SjAb, followed by the silver enhancement process, dissolution of silver metal atoms in an acidic solution, and determination of dissolved silver ions by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. Assay conditions were optimized, including the reaction time of SjAg with SjAb, the interaction of SjAb with the colloidal gold-labeled secondary antibody, the dilution ratio of the colloidal gold-labeled secondary antibody and the silver enhancement time. The integration of the anodic stripping peak current depended linearly on the SjAb logarithmic concentration over the range of 6.4 ng/ml to 100 μg/ml. A detection limit as low as 3.0 ng/ml SjAb was achieved, which was better than the piezoelectric body acoustic wave sensor (detection limit of 7.2 μg/ml) and the renewable amperometric immunosensor (detection limit of 0.36 μg/ml). Rabbit serum samples with various degrees of infection were analyzed, and the results demonstrate that the proposed method meets the requirements of clinical analysis.

Electron microscopic immunogold localization of salivary mucins MG1 and MG2 in human submandibular and sublingual glands.

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.

Identification and localization of an adhesin on the surface of Tritrichomonas foetus.

Tritrichomonas foetus is a mucosal parasite of the urogenital-vaginal tract of cattle that strongly adheres to erythrocytes, which suggests that it presents an adhesin that recognizes red blood cells from different animal species and blood groups. In the present report we describe a cell-fractionation method for obtainment of a membrane fraction of T. foetus, which adhered to red blood cells. The T. foetus adhesin was obtained after parasite lysis and fractionation followed by ultracentrifugation, whereby a 100,000-g pellet fraction showed a strong hemagglutinating activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction, of erythrocyte ghosts, and of ghosts allowed to interact with the parasite membrane fraction revealed the presence of a 100-kDa protein as the putative adhesin. Polyclonal antibodies obtained in rabbits immunized with this protein recognized proteins of 100 and 90 kDa as determined by immunoblotting. Confocal laser scanning microscopy and transmission electron microscopy of cells incubated first in the presence of the antibody and subsequently in the presence of fluorescein- or gold-labeled goat anti-rabbit IgG showed labeling of the protozoan surface as well as of some cytoplasmic vesicles.

Submitochondrial Distribution of Three Key Steroidogenic Proteins (Steroidogenic Acute Regulatory Protein and Cytochrome P450scc and 3β-Hydroxysteroid Dehydrogenase Isomerase Enzymes) upon Stimulation by Intracellular Calcium in Adrenal Glomerulosa Cells

In adrenal glomerulosa cells, angiotensin II (Ang II) and potassium stimulate aldosterone synthesis through activation of the calcium messenger system. The rate-limiting step in steroidogenesis is the transfer of cholesterol to the inner mitochondrial membrane. This transfer is believed to depend upon the presence of the steroidogenic acute regulatory (StAR) protein. The aim of this study was 1) to examine the effect of changes in cytosolic free calcium concentration and of Ang II on intramitochondrial cholesterol and 2) to study the distribution of StAR protein in submitochondrial fractions during activation by Ca2+ and Ang II. To this end, freshly prepared bovine zona glomerulosa cells were submitted to a high cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nM) for 2 h. Mitochondria were isolated and subfractionated into outer membranes, inner membranes (IM), and contact sites (CS). Stimulation of intact cells with Ca2+ or Ang II led to a marked, cycloheximide-sensitive increase in cholesterol in CS (to 143 +/- 3. 2 and 151.1 +/- 18.1% of controls, respectively) and in IM (to 119 +/- 5.1 and 124.5 +/- 6.5% of controls, respectively). Western blot analysis revealed a cycloheximide-sensitive increase in StAR protein in mitochondrial extracts of Ca2+-clamped glomerulosa cells (to 159 +/- 23% of controls). In submitochondrial fractions, there was a selective accumulation of StAR protein in IM following stimulation with Ca2+ (228 +/- 50%). Similarly, Ang II increased StAR protein in IM, and this effect was prevented by cycloheximide. In contrast, neither Ca2+ nor Ang II had any effect on the submitochondrial distribution of cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase isomerase. The intramitochondrial presence of the latter enzyme was further confirmed by immunogold staining in rat adrenal fasciculata cells and by immunoblot analysis in MA-10 mouse testicular Leydig cells. These findings demonstrate that under acute stimulation with Ca2+-mobilizing agents, newly synthesized StAR protein accumulates in IM after transiting through CS. Moreover, our results suggest that the import of StAR protein into IM may be associated with cholesterol transfer, thus promoting precursor supply to the two first enzymes of the steroidogenic cascade within the mitochondria and thereby activating mineralocorticoid synthesis.

Immunogold electron microscopic localization of phosphoenolpyruvate carboxykinase in rat liver

Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-limiting gluconeogenic enzyme which catalyzes the conversion of oxaloacetate to phosphoenolpyruvate. A gradient of PEPCK from periportal hepatocytes to pericentral hepatocytes has been demonstrated in normal rat liver; however, the subcellular distribution of PEPCK molecules within hepatocytes could not be determined from these light microscope studies.We have used two immunogold histochemical approaches to study the subcellar distribution of PEPCK in normal rat hepatocytes. In the first procedure 10μm cyrosections of 4% paraformaldehyde perfusion-fixed rat liver were collected on silanated slides and immunostained with goat anti-rat PEPCK (normal goat serum served as a control), and incubated in a secondary antibody (5nm gold-labeled rabbit anti-goat IgG) and a tertiary antibody (5nm gold-labeled goat anti-rabbit IgG). The immunogold stained samples, with or without silver enhancement (IGSS), were counterstained with pyronine Y, re-embedded in Visio- Bond and removed from the slides and semithin sectioned for epipolarized illumination combined with transmitted light microscopy (Figs. 1 and 2), or re-embedded in epoxy resin and sectioned for electron microscopy (EM, Fig. 4) In the second procedure 25, 50 and 100 μm free-floating vibratome sections of well fixed rat liver (4% paraformaldehyde plus 0.5% glutaraldehydeperfusion-fixed, immersion fixed overnight at 4°C) were immunogold stained using a 5nm gold labeled secondary antibody, with and without IGSS, osmicated, embedded in epoxy resin and sectioned for EM (Figure 3).

A new method for transfer of polyethylene glycol-embedded tissue sections to silanated slides for immunocytochemistry.

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.

Topographical relationships between catecholamine-and neuropeptide-containing fibers in the median eminence of the newt,Triturus alpestris

Dopaminergic and peptidergic nerve fibers were simultaneously demonstrated with a double-labeling technique at the ultrastructural level. The first antibody, raised against tyrosine hydroxylase, was applied during the preembedding phase and visualized with the peroxidase method. The second antibody, raised against one of the peptides met-enkephalin, somatostatin or gonadotropin-releasing hormone (GnRH), was applied to the ultrathin sections and visualized with gold-labeled goat anti-rabbit IgG. The fibers of both categories were present in the zona externa of the median eminence, frequently contacting the basal lamina of the portal vessels. In addition, topographical relationships between different types of nerve fibers were observed in the perivascular areas, although there were no morphological signs of synaptic specializations. Using serial sections, it could be established that one GnRH-fiber contacted both a dopaminergic fiber and a fiber immunoreactive for met-enkephalin. The observations support earlier physiological data concerning the regulation of the hypothalamo-hypophyseal axis, with special emphasis on the release of neurohormones in the median eminence of the newt.

Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy.

We used post-embedding immunocytochemical techniques and affinity-purified anti-actin antibody to evaluate localization of actin in epithelial cells of small intestine by fluorescence and electron microscopy. Small intestine was fixed with 2% formaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M. One-micron or thin sections were stained with antibody followed by rhodamine- or colloidal gold-labeled goat anti-rabbit IgG, respectively. Label was present overlying microvilli, the apical terminal web, and the cytoplasm directly adjacent to occluding and intermediate junctions. Label was associated with outer mitochondrial membranes of all cells and the supranuclear Golgi region of goblet cells. Lateral cytoplasmic interdigitations between mature cells and subplasmalemmal filaments next to intrusive cells were densely labeled. The cytoplasm adjacent to unplicated domains of lateral membrane was focally labeled. Label was prominent over organized filament bundles within the subplasmalemmal web at the base of mature cells, whereas there was focal labeling of the cytoplasm adjacent to the basal membrane of undifferentiated cells. Basolateral epithelial cell processes were labeled. Label was focally present overlying the cellular ground substance. Our results demonstrate that actin is distributed in a distinctive fashion within intestinal epithelial cells. This distribution suggests that in addition to its function as a structural protein, actin may participate in regulation of epithelial tight junction permeability, in motile processes including migration of cells from the crypt to the villus tip, in accommodation of intrusive intraepithelial cells and in adhesion of cells to one another and to their substratum.

Peripheral vesicles in proplastids of barley stripe mosaic virus-infected wheat cells contain double-stranded RNA

Cytochemical and immunoelectron microscopy localized dsRNA in root tips systemically infected with barley stripe mosaic virus (BSMV). Anti-polyinosinic:polycytidylic acid (Anti-poly(I):poly(C)) serum was used as a primary stain followed by gold-labeled goat anti-rabbit IgG complexes. Only vesicles of vesiculated proplastids of early-infected cells but not proplastids of cells from noninoculated plants, contained dsRNA. This supports the notion of a role for proplastids in BSMV replication.

Immunocytochemistry in plants with colloidal gold conjugates

The chitin-binding lectin wheat germ agglutinin (WGA) is found at the periphery of wheat embryos, and a similar lectin is present at the root tips of older plants (Mishkind et al. 1982). Although a ferritin-conjugated secondary antibody is adequate for localizing WGA in embryos, native electron-opaque particles make the electron microscope identification of added label equivocal in other wheat tissues. As reported here, however, unambiguous ultrastructural localization of WGA-like lectin in adult wheat roots can be obtained with rabbit anti-WGA followed by colloidal gold-labeled goat anti-rabbit (GAR) IgG. Colloidal gold (CG) was prepared by the reduction of gold chloride with citrate, ascorbate or phosphorous. GAR IgG, prepared from serum by antigen affinity chromatograhy, was adsorbed to the gold particles to produce a stabilized suspension of GAR-CG. Localization was performed on 8–12 μM frozen sections of tissue fixed in 4% paraformaldehyde, 0.3% glutaraldehyde, and 0.75% acrolein in phosphate-buffered saline containing 1M sucrose. Localization with GAR-CG was first compared to that ascertained in embryos using other probes and was then extended to the roots of adult plants. An advantage of the GARCG method is that it permits the visualization of antigen at both the light and electron microscope levels in the same section. At the light level, the anti-WGA-GAR-CG complex appears as a red stain that is localized in specific tissues of embryos and in the caps and outer layers of adult roots. Sections in which lectin was detected at the light microscope level were embedded in plastic and sectioned for subcellular examination. Electron dense gold particles indicative of WGA are found at the periphery of protein bodies in wheat embryos and in vacuoles of the roots of adult plants. Sections incubated with control IgG lack reaction product.

Immunohistochemical localization of barley stripe mosaic virions in infected wheat cells

Barley stripe mosaic virus particles were localized in ultrathin sections with colloidal gold-labeled specific IgG or antiserum followed by gold-labeled goat anti-rabbit IgG. On the average, 1.5 gold particles were attached per virus rod. A statistical analysis of counts of gold and virus particles showed that the staining procedure was highly reproducible from experiment to experiment and after several independently prepared colloidal gold solutions. The procedure should be useful for the intracellular localization of any protein to which an antibody can be prepared.