A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens

Summary In order to study the morphological interrelationships between immunocytochemically identified neuronal systems, a double labelling procedure — suitable for correlative light and electron microscopic observations — is introduced. The technique is based on the consecutive use of the silver-gold (SG) intensified and non-intensified forms of the oxidized 3,3-diaminobenzidine (DAB) chromogen in the framework of the peroxidase-antiperoxidase complex (PAP) indirect immunocytochemical procedure. The first tissue antigen is detected by the SG intensified DAB chromogen, which has a black color and high electron density. The structures containing the second antigen are visualized by the non-intensified DAB-endproduct, which is less electron dense than the silver-gold amplified form and is brown. The metallic shield that forms around the labeled antibody sequences associated with the first antigen prevents non-specific binding of immunoglobulins used for the detection of the second tissue antigen.The application of this method for the simultaneous detection of tyrosine hydroxylase (TH)- and corticotropin releasing factor (CRF)-immunoreactive structures revealed that black colored TH-immunopositive fibers contacted brown colored CRF-synthesizing neurons in the hypothalamic paraventricular nucleus. The juxtaposition of TH-and CRF-containing elements was apparent in both thick vibratome (40 m) and semithin (1 m) sections. At the ultrastructural level, TH-positive terminals — labeled by silvergold grains — were observed to establish asymmetric synapses with both CRF- and TH-immunoreactive neurons. The former finding indicates a direct, TH-immunopositive, catecholaminergic influence upon the hypothalamic CRF system, while the latter demonstrates the existence of intrinsic connections between TH-positive elements.Supported by NIH Grant NS19266     

S100A6 and S100A11 are specific targets of the calcium- and zinc-binding S100B protein in vivo

In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.     

Histochemical demonstration of sugar residues by lectin and immunocytochemical techniques for blood group antigens in human colon

Summary Goblet cell mucin in 39 human colons was studied by methods specific for various sugar residues, including staining with three lectins,Dolichos biflorus agglutinin (DBA, specific for blood group A antigen),Griffonia simplicifolia agglutinin-I (GSA-I, B) and peanut agglutinin (PNA, T antigen), and immunostaining for A, B, H and T. Isoantigens A, B or H were found only in the right colon. GSA-I reactive goblet cells occurred in the right colon of both blood group A and B patients and possibly contained isoantigens. However DBA reactive cells were found in all cases. Prior neuraminidase digestion imparted anti-A, GSA-I and DBA reactivities to the cells lining the lower crypts in all cases. This pretreatment also imparted PNA and anti-T reactivities to goblet cells, only the latter reactivity being eliminated by galactose oxidase. Goblet cell mucin in transitional mucosa revealed decreased A and B, and increased H antigens. Enhanced galactose oxidase—Schiff (GOS) and anti-T reactivities were also noted. The present results revealed that some lectin reactions of goblet cells might be related to blood group antigens but others were not, and that different techniques for demonstrating reputedly the same sugar residues produced different results, indicating a need for proper evaluation of their specificity.     

Multimarker immunohistochemical staining of calgranulins, chloroacetate esterase, and S100 for simultaneous demonstration of inflammatory cells on paraffin sections

Mac387 monoclonal antibody (MAb) recognizes two calcium binding, myeloid-associated proteins, now termed calgranulins, expressed at high levels by neutrophils and monocytes. Calgranulins are related to migration inhibitory factor (MIF) and are lost in a few days from monocytes differentiated in vitro. This marker is therefore potentially useful to analyze macrophage heterogeneity and turnover in tissue sections. In this study, we developed an immunohistochemical multimarker technique, including calgranulin demonstration, suitable for analyzing different inflammatory cells on paraffin-embedded material. The technique was carried out in subsequent steps demonstrating (a) naphthol AS-D chloroacetate esterase (CAE); (b) S100 immunoreactivity using a rabbit antibody in peroxidase-antiperoxidase (PAP) staining; and (c) Mac387 immunoreactivity using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. CAE staining was introduced in this method to distinguish Mac387+/CAE- macrophages from Mac387+/CAE+ neutrophils, and Mac387-/CAE+ mast cells. S100 protein is strongly expressed within lymphoid tissues by dendritic accessory cells and was then applied to distinguish these cells from S100-macrophages. We have also verified the possibility of reducing the staining time for this time-consuming procedure by use of microwave irradiation. The technique was applied to a representative variety of normal and pathological samples to assess its usefulness for study of cell heterogeneity. Our results showed the multimarker technique to be highly informative in the study of inflammatory lesions (e.g., rheumatoid arthritis, sarcoid and cat-scratch granulomas, dermathopathic lymphadenopathy), and is of wide potential value as an aid to histopathological diagnosis of several diseases.     

A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity.

We describe a fluorescent histochemical technique for detection of nonspecific alkaline phosphatase (APase) in cells. The technique utilizes standard azo dye chemistry with naphthol AS-MX phosphate as substrate and fast red TR as the diazonium salt. The reaction product is a highly fluorescent red precipitate. Pre-implantation mouse embryos were used to establish optimal fixation and staining protocols and the specificity and sensitivity of the method. Fixation was in 4% paraformaldehyde for 1 hr, as glutaraldehyde induced autofluorescence of the cells. Maximal discriminable staining was detected after 15-20 min in the stain solution. The stain solution itself proved to be non-fluorescent, thus allowing visual observation of the progress of the staining reaction by fluorescence microscopy in its presence. To test the specificity of this fluorescent APase stain, a variety of cell types of known APase reactivity were stained by this protocol. Mouse lymphocytes and STO fibroblasts were negative, whereas F9 teratocarcinoma cells, intestinal epithelial cells, and rat fetal primordial germ cells were all found to be highly positive for APase activity, in agreement with published results on APase localization in these cells.     

A new technique for simultaneous collection of macroalgal propagules in the water column

A Simultaneous Collection Module (SCM) was designed to concurrently collect multiple samples of macroalgal propagules, and to evaluate their abundance and spatial distribution in the water column. The basic sampling units are water collectors (tubes), held together to form an evenly spaced grid forming a spatial array with three dimensions. Each collector in the module possesses a simultaneous closing mechanism. The water collected in each tube is then filtered and the propagules retained on each filter cultivated under controlled conditions. The sampling system was deployed in the field in central Chile. Results suggest the system is an efficient collector, able to provide data on time-space changes of macroalgal propagules in the water column.     

S100A6 - New facts and features

S100A6 (calcyclin) is a 10.5 kDa Ca²⁺-binding protein that belongs to the S100 protein family. S100A6 contains two EF-hand motifs responsible for binding of Ca²⁺. It also binds Zn²⁺ through not yet identified structures. Binding of Ca²⁺ induces a conformational change in the S100A6 molecule which in consequence increases its overall hydrophobicity and allows for interaction with target proteins. S100A6 was found in different mammalian and avian (chicken) tissues. A high level of S100A6 is observed in epithelial cells, fibroblasts and in different kinds of cancer cells. The function of S100A6 is not clear at present, but it has been suggested that it may be involved in cell proliferation, cytoskeletal dynamics and tumorigenesis. Additionally, S100A6 might have some extracellular activities. This review presents new facts and features concerning the S100A6 protein.     

Immunohistochemical localization of S100A1 and S100A6 in postnatally developing salivary glands of rats

 S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0–5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands. Accepted: 14 July 1998     

A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling.

We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.     

A new direct lead technique for histochemical demonstration of alkaline phosphatase activity.

A lead method for demonstrating alkaline phosphatase is described. The method is based on direct precipitation of lead as lead phosphatase at pH 9.5, the pH optimum of the enzyme. Stable incubation medium was achieved by using tartrate, instead of maleate, as chelating for lead. The method was found to be suitable for visualization of alkaline phosphatase in different types of tissues.     

In situ chromosome preparation technique for simultaneous cytogenetic and immunocytochemical studies on cell cultures of solid tumors

Summary Immunophenotyping of cultured cancer cells requires intact antigenic structures; these are mostly destroyed by conventional chromosome preparation techniques. Thus, the simultaneous cytogenetic and immunocytochemical characterization of solid tumor cells appears unfeasible. Here, we describe a novel method that allows in situ chromosome preparation from monolayer cultures of solid tumor cells without affecting their immunological features. Using this technique, it is possible to achieve detailed cytogenetic data including chromosome banding together with the demonstration of cytoplasmic and nuclear antigens within the same tumor cell.     

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.     

Demonstration of Heterodimer Formation between S100B and S100A6 in the Yeast Two-Hybrid System and Human Melanoma

S100B (S100β) and S100A6 (calcyclin) are two 10-kDa Ca²⁺- and Zn²⁺-binding proteins coexpressed in melanoma and cell-cycle regulated. These proteins are members of the S100 subfamily and are thought to exert their function through interaction with intracellular target proteins. In order to search for potential target proteins interacting with S100B, we used a yeast two-hybrid strategy with human S100B as bait to screen a human brain cDNA library. The fusion proteins interacting with the S100B bait were identified as S100B, S100A1, and S100A6. This indicates the potential of S100B to form homodimers and heterodimers with other members of the S100 subfamily. By Northern and Western blotting, S100B and S100A6 were shown to be expressed at high levels in a panel of human melanoma cell lines. S100B and S100A6 were coimmunoprecipitated from melanoma cell lysates in the presence of 100 μM Zn²⁺. Confocal microscopy demonstrated that both proteins were distributed throughout the cytoplasm and concentrated in the nucleus. The demonstration of an association and colocalization of S100B and S100A6 in melanoma supports the possibility that an S100B/S100A6 heterodimer plays a functional role in these cells.     

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.