Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.     

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35–1.0 m) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 m. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.     

Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy

We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.     

An investigation of optimal gold particle size for immunohistological immunogold and immunogold-silver staining to be viewed by polarized incident light (EPI polarization) microscopy

We investigated the optimal gold particle size for use with polarized incident light (epi polarization) microscopy with immunogold immunohistological preparation in both immunogold indirect (IGS) and silver-enhanced immunogold-silver staining (IGSS) techniques. A range of gold particle sizes from 5 nm-40 nm was used along with tissue of known immunoreactivity with a well-characterized primary monoclonal antibody. Checkerboard titrations were carried out for each technique and for each particle size. The preparations were viewed using a standard polarized incident light microscope and assessed in a semi-quantitative manner. Adequate visualization of gold particles was achieved using the indirect staining method only with a particle size of 40 nm. With silver enhancement (IGSS), particles of all sizes were clearly seen. However, 5-nm particles were considered optimal for this method because of reduced background staining, high titration of antisera possible, and crisp localization of the visual signal.     

Immunogold labeling of metaphase cells

A method is presented for the phenotypic identification of metaphase cells stained for chromosome aberration and SCE analysis. The cells are labeled in suspension with antibodies conjugated with colloidal gold, and then chromosome preparations are made using a cytocentrifuge. A silver development (IGSS) procedure is used to enhance the gold labeling for light microscopy. A variety of fixatives may be employed, permitting various cytogenetic and cytochemical staining procedures to be used.     

抗体和寡核苷酸双标记纳米金生物探针的制备及性能分析

基于纳米金粒子与抗体静电吸附作用,与硫醇修饰的寡核苷酸共价结合,建立一种新的双标记纳米金生物探针的制备方法.通过透射电镜（TEM）、紫外光谱、斑点免疫金渗滤法、免疫金银染色光镜观察法、荧光标记法等检测探针表征,及表面抗体活性情况和寡核苷酸的覆盖率,同时采用变性聚丙烯酰胺凝胶电泳（PAGE）检测寡核苷酸的存在.结果表明,纳米金粒子同时连接抗体和寡核苷酸后生物性能良好,且每个纳米金粒子（10±3）nm表面可覆盖寡核苷酸(92±20)条,双标记纳米金生物探针的制备具有简捷、稳定的特点.可作为一种新型探针应用于超微量蛋白质检测.     

Electron microscopic in situ DNA nick end-labeling in combination with immunoelectron microscopy.

We describe an in situ DNA nick end-labeling method that can be performed at the electron microscopic level and can also be combined with immunoelectron microscopy. As the materials, we used skin tissues from normal skin and from Bowen's disease that had been cryofixed, freeze-substituted, and embedded in Lowicryl K11M resin. Ultrathin sections were cut and incubated with a reaction buffer containing digoxigenin-dUTP and terminal deoxynucleotidyl transferase. Digoxigenin nucleotides were labeled with anti-digoxigenin antibodies conjugated with colloidal gold. Specific signals were detected in the condensed chromatin of differentiated epidermal cells and hair follicles in normal skin and of dyskeratotic cells in Bowen's disease. The labeling density over chromosomal areas of apoptotic cells was significantly higher than that over chromosomal areas of mitotic cells or cytoplasmic areas. Ultrastructure was well preserved and double staining with an anti-keratin antibody was also successfully performed. This simple method has a wide range of applications to identify the nature of apoptotic cells and explore the mechanisms of apoptosis.     

Comparison of detecting sensitivities of different sizes of gold particles with electron-microscopic immunogold staining using atrial natriuretic peptide in rat atria as a model

The detecting sensitivities of different-sized gold particles were compared in the localization of atrial natriuretic peptide (ANP) in rat atria. The secondary antibodies were goat antirabbit labeled with 5, 15, 30, or 40 nm colloidal gold diluted 1:2 to 1:100 in Tris buffer. The relative quantity of alpha-ANP immunoreactivity in specific granules was determined by subtracting the number of gold particles in 1 micron 2 nongranule area from that in 1 micron 2 granule area measured with a computerized image analyzer. The optimal dilution that achieved the maximal contrast between specific and background label was influenced by the particle size. Optimal dilutions were 1:80, 1:30, 1:20, and 1:5 for 5, 15, 30, and 40 nm gold, respectively. At optimal dilutions, the maximal detecting sensitivity (MDS) was in inverse proportion to the gold particle size; however, this relationship is not entirely linear. The ratio among the MDSs of 5, 15, 30, and 40 nm gold particles was approximately 34:9:3:2. A double immunogold staining was performed to localize alpha- and beta-ANPs with 15 and 5 nm gold, respectively. Both antigens were detected in the same granules. If the ratios established from the single staining data were used, the ratio between the alpha- and the beta-ANP antigens in the same granules was approximately 2.8:1. The data obtained in this study provide a useful reference for applications of immunogold electron microscopy in a quantitative manner, particularly for double immunogold labeling.     

Immunocytochemical localization of cathepsin H in rat kidney. Light and electron microscopic study

Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For light microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Immunocytochemical localization of cathepsin H in rat kidney

Summary Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For ligh microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

一种酚氧化酶原组织定位的胶体金标记免疫电镜方法

以亚洲玉米螟Ostrinia furnacalis Guenée5龄幼虫的中肠和体壁组织为材料,采用Epon812常规包埋方法,以作者实验室制备的酚氧化酶原多克隆抗体为一抗、胶体金标记的羊抗鼠IgG为二抗,采用醋酸双氧铀-柠檬酸铅双染色体系,建立一种胶体金标记的特异性强且超微结构保存较好的亚洲玉米螟幼虫体内中酚氧化酶原免疫电镜定位方法。     

BrdUrd labeling of S-phase cells in testes and small intestine of mice, using microwave irradiation for immunogold-silver staining: an immunocytochemical study.

In this study, BrdUrd labeling of S-phase cells in the small intestine and testes was accomplished using microwave irradiation. In this way crypt cells, spermatogonia, and Leydig cells could be labeled using removable plastic-embedded sections and immunogold-silver staining (IGSS). By using short periods of microwave irradiation for incubation of the monoclonal antibodies and the protein A-colloidal gold solution, the detection of BrdUrd-labeled cells could be remarkably enhanced. A comparative study of BrdUrd labeled spermatogonia in the testis of a Cpb-N mouse that received both [3H]-thymidine and BrdUrd proved that 90% of the BrdUrd-labeled cells also showed [3H]-thymidine labeling. The radioactive [3H]-thymidine labeling was a time-consuming method of 4 weeks' duration, whereas the BrdUrd-labeled cells could be labeled, fixed, enhanced, and counterstained in less than 3 hr. This investigation proves that BrdUrd labeling of S-phase cells can be a reliable, reproductive, rapid, and non-radioactive alternative method for [3H]-thymidine labeling of proliferating cells.     

Microwave-aided technique to detect bromodeoxyuridine in S-phase cells using immunogold-silver staining and plastic-embedded sections

Summary A technique is described to detect bromodeoxyuridine (BrdU) incorporate by cells in S-phase, with a monoclonal antibody, using removable plastic embedding and immunogold-silver staining (IGSS). The incubation times were reduced and the immunological reactions enhanced by microwave irradiation.The embedding in methyl methacrylate enabled us to make thinner sections and it improved the quality of the preparations. The methyl methacrylate did not hinder the reaction of BrdU with the antibody because it could be removed prior to the IGSS procedure. The IGSS procedure appeared to be very sensitive, requiring lower concentrations of the antibodies than other methods. The use of microwave irradiation shortened the time needed to stain a section from 7 to less than 4 h. Furthermore, using microwave irradiation, the concentration of the antibodies needed could be reduced even further compared with the normal IGSS procedure.In sections of the mouse testis and small intestine only nuclei of cells known to be able to proliferate appeared BrdU positive. The non-specific background staining was found to be negligible. In testes of mice that received both³H-thymidine and BrdU more than 95% of the radioactively labelled cells also showed BrdU label and vice versa. This indicates that both methods are equally sensitive for detecting cells in S-phase.     

The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.

We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.     

Ultrastructural immunocytochemical localization of 5-hydroxytryptamine in gastric enterochromaffin cells

Enterochromaffin (EC) cells in the gastrointestinal tract are known to contain 5-hydroxytryptamine (5HT). The probable ultrastructural localization of 5HT in the dense core vesicles ( DCVs ) of EC cells is based on the use of histochemical techniques, such as argentaffinity and the potassium dichromate reaction. In the present paper we describe an immunocytochemical method for specifically localizing 5HT in EC cells by electron microscopy. Pieces of mucosa from the pyloric region of the rabbit stomach were prepared for electron microscopy by fixation in 0.5% glutaraldehyde-picric acid-formaldehyde without osmication , and then embedded in LX-112. Thick sections (1 micron) were mounted on glass slides and processed for the fluorescence immunocytochemical localization of 5HT. Thin sections (60-90 nm) were mounted on formvar-coated slot grids and processed for the ultrastructural immunocytochemical localization of 5HT. Both the thick and thin sections were processed by an identical procedure, beginning with a 30-min incubation in anti-5HT antiserum diluted 1:1400, followed by an IgG-FITC-gold-labeled second antibody. Fluorescent EC cells were consistently observed in the thick sections of gastric mucosa. By carefully trimming and sectioning the adjacent block face, the identical EC cell could be identified by electron microscopy. A quantitative analysis revealed the number of gold particles in EC cells to be significantly greater over the cores of DCVs than over the non-core cytoplasm or over the nucleus. Absorption of the primary antiserum with 5HT abolished all labeling, while absorption with a 5HT precursor, 5-hydroxytryptophan, did not significantly reduce core labeling. Non-EC epithelial cells were not labeled. These results demonstrate that immunoreactive 5HT in EC cells is stored in the cores of DCVs .     

STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein

Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of ～300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.     

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.     

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.     

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     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.