Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry

Summary A method was developed that allows the analysis of neuropeptides and monoamines in a single tissue section by the application of the unlabeled antibody method for peptide staining to tissue sections freeze-dried for formaldehyde-induced monoamine histofluorescence. The hypothalamic magnocellular system of male albino rats served as a model for this study; neurons were stained with anti-neurophysin sera, which mark the vasopressin- and oxytocin-associated proteins. Neurophysin-containing perikarya appeared to be surrounded by catecholamine-containing varicosities. This phenomenon was seen to varying degrees within the supraoptic and paraventricular nuclei. The juxtaposition of varicosities and peptidergic neurons suggests an afferent fiber-target neuron relationship that might favor a functional interaction between monoamines and neuropeptides.Supported by USPHS Grant and postdoctoral NS15816 (JRS) fellowship AG01575 (THM)     

Quantitative ultrastructural immunocytochemistry using a computerized image analysis system

Secretory granules in human pituitary adenoma cells have been examined indirectly for hormone epitopes by immunogold labelling of resin-embedded ultrathin sections. The specific binding of different immunoglobulin-gold complexes to the antigrowth hormone antibodies over the secretory granules was measured using a computerized image analysis system. This facilitated the assessment of the preferential binding to the target granules of gold particles with three different average particle diameters (Au7, Au11, Au17). The time of pretreatment of sections with H2O2 or a buffer was found to influence the staining considerably. The scanning electron microscopic findings of protruded secretory granules with a mountain-like surface might be relevant to the uneven distribution of immunolabels seen over the secretory granules in the adenohypophysis.     

Detection of plasma cell immunoglobulins in tissue sections optimally fixed for ultrastructural immunocytochemistry

We describe the ultrastructural localization of plasma cell immunoglobulins in vibratome sections of popliteal lymph nodes. Fixation with glutaraldehyde-paraformaldehyde gave better tissue and antigen preservation than paraformaldehyde or periodic acid lysine-paraformaldehyde; biotinylated Fab fragments of sheep anti-mouse IgG-streptavidin-biotinylated horseradish peroxidase (HRP) or Fab-HRP conjugates gave similar results. With both immunoreagents, excellent tissue preservation and antigen detection was observed in the first layer of cells sectioned with the vibratome. Conjugates of anti-mouse IgG with HRP did not show any staining. Peroxidase stain was observed in the nuclear envelope, cisternae of the rough endoplasmic reticulum, and the Golgi apparatus complex. In the Golgi apparatus, staining was seen consistently in cisternae of the cis face and in adjacent vesicles; the trans cisternae showed weak or no stain, and adjacent vesicles, "coated" vesicles, and granules were not stained. This study shows that high quality of tissue preservation and antigen detection, by both light and ultrastructural immunocytochemistry, is feasible in tissue fixed with glutaraldehyde-paraformaldehyde followed by vibratome sectioning and immunostaining with Fab-biotin-streptavidin-biotin-HRP, or Fab-HRP.     

Simultaneous detection of multiple single-base alleles at a polymorphic site.

PCR amplification of multiple specific alleles (PAMSA) is a rapid method of detecting single-base polymorphisms. We demonstrate its utility by detecting a polymorphism in an Alu sequence in the factor IX gene.     

Studies of hCG binding and endocytosis in rat ovary by ultrastructural immunocytochemistry

Summary Localization of hCG binding sites and the process of endocytosis in pseudopregnant rat ovaries were investigated by indirect electron-microscopic immunocytochemistry. Immature female rats were treated with pregnant-mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce ovarian luteinization. Eight days after priming with PMSG-hCG and 1–6 h before sacrifice the animals were given another injection of hCG to bind the receptors. Receptor sites to hCG localized by reaction product were present in most luteal cells, but not in primary follicular cells. The receptor sites were distributed on luteal cell surfaces facing interstitial spaces. Endocytotic pits containing hCG binding sites were rarely seen 1 h after hCG injection. At 2 h, hCG and presumably its receptor were taken up within endocytotic vesicles with the evidence of reaction product coated on the vesicle wall. With time, fusion of endocytotic vesicles with lysosome occurred and the reaction product appeared in phagolysosomes. The reaction product was localized on phagolysosomal inner surface or in free granular form. These findings suggest that hCG and its receptors were internalized through endocytotic pits and endocytotic vesicles and delivered to lysosomes probably for degradation. An additional experiment for localization of acid phosphatase was also performed to delineate the lysosomes and phagolysosomes.     

Posttranslational isomerization of a neuropeptide in crustacean neurosecretory cells studied by ultrastructural immunocytochemistry

Isomerization of the third amino acid residue (a phenylalanine) of crustacean hyperglycemic hormone (CHH) has been previously reported to occur as a late step of hormone precursor maturation in a few neurosecretory cells in the X-organ-sinus gland complex of the crayfish Orconectes limosus. In the present report, using conformation-specific antisera combined with immunogold labeling, we have studied, at the ultrastructural level, the distribution of L- and D-CHH immunoreactivity in CHH-secreting cells of the crayfish Astacus leptodactylus. Two CHH-secreting cell populations were observed, the first one (L-cells), the most numerous, exhibited only labeling for L-CHH. In the second one (D-cells), four secretory granule populations were distinguished according to their labeling: unlabeled, either L- or D- exclusively or both L- and D-granules. Labeling quantification by image analysis in D-cells showed a marked increase in D-labeling from the cell body to the axon terminal. However some L- and mixed granules remain in axon terminals. Our results demonstrate that Phe3 isomerization of CHH occurs within the secretory granules of specialized neurosecretory cells and progresses as the granules migrate along the axonal tract. The observation that not all the CHH synthesized is isomerized, and the great variability in the proportion of L- and D-immunoreactivity in granules in every cell region may suggest an heterogeneous distribution of the putative enzyme involved in Phe3 isomerization, a peptide isomerase, within the secretory pathway.     

Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.

We have modified our previous method for immunogold staining of unosmicated, plastic-embedded tissue by addition of tannic acid as a post-fixative to increase membrane contrast. Overall cell ultrastructure and organelle membranes, in particular, appeared well preserved after this treatment. We evaluated quantitatively the effect of tannic acid on the antigenicity of several membrane proteins in rat liver and intestine. For all antigens tested, significant antigenicity was retained on both intracellular and plasma membranes. However, the level of antigenicity decreased with increased concentrations of tannic acid. This effect was most apparent on the apical and basolateral membranes of hepatocytes and on the apical membrane of enterocytes, surfaces that had been in direct contact with the tannic acid fixative. The results indicate that when low concentrations of tannic acid are employed, this method yields greatly enhanced membrane contrast while preserving sufficient antigenicity to facilitate the ultrastructural localization of many membrane and other antigens.     

Extracellular matrix components of the placental extravillous trophoblast: immunocytochemistry and ultrastructural distribution

 Invasive extravillous trophoblast cells of the human placenta are embedded in a self-secreted extracellular matrix, the matrix-type fibrinoid. The ultrastructure and molecular composition of the matrix-type fibrinoid of the term human placenta were studied by transmission electron microscopy and immunogold labelling. We used antibodies directed against different matrix proteins such as collagen type IV, laminin, vitronectin, heparan sulfate, various fibronectin isoforms, and against the oncofetal blood group antigen, ”i”. Immunogold labelling patterns of matrix proteins are the basis for the subdivision of the trophoblast-derived matrix-type fibrinoid into mosaic-like patches of structurally and immunocytochemically different compartments. Firstly, fine granular patches with structural similarities to basal lamina material are composed solely of collagen type IV and laminin. Secondly, an ultrastructurally amorphous glossy substance shows reactivity with antibodies against heparan sulfate and vitronectin. A third type of patches, fine fibrillar networks embedded in the above-mentioned glossy matrix, are reactive with antibodies against normal fibronectin isoforms (IST-4, IST-6, IST-9) and oncofetal isoforms (BC-1, FDC-6). The blood group precursor antigen ”i” was not only expressed on the surfaces of the extravillous trophoblast cells but was associated with the fibronectin-positive fibrils. In conclusion, within this extracellular matrix, clear compartments of different composition can be distinguished from each other. Glycosylation with ”i” in this matrix may be involved in immunological masking, thus preventing rejection of placenta and fetus. Accepted: 6 May 1996     

Simultaneous and sensitive detection of dual DNA targets via quantum dot‐assembled amplification labels

We describe a signal amplification assay for the simultaneous detection of HIV‐1 and HIV‐2 via a quantum dot (QD) layer‐by‐layer assembled polystyrene microsphere (PS) composite in a homogeneous format. The crucial point of this composite is the core–shell system. PS is utilized as the core and QDs as the shell. Based on the high affinity of streptavidin and biotin, QDs are assembled layer‐by‐layer on the surface of the PS as amplification labels. Biotinylated reporter probe is combined with the PS–QDs conjugate and then hybridized with target DNA immobilized on the surface of a 96‐well plate. Using this approach, each target DNA corresponds to a large number of QDs and the fluorescence signal is greatly enhanced. Two QD colors (605 and 655 nm) are used to detect dual‐target DNAs simultaneously. Taking advantage of the enzyme‐free reaction and high sensitivity, this PS–QD‐based sensor can be used in simple ‘mix and detection’ assays. Our results show that this technology has potential application in rapid point‐of‐care testing, gene expression studies, high‐throughput screening and clinical diagnostics. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of pancreatic glucagon cells in the cartilaginous fish Scyliorhinus canicula by ultrastructural immunocytochemistry

The ultrastructural localization of glucagon in the presence of Scyliorhinus canicula was investigated. We used a post-embedding immunoelectron microscopy method on pancreatic samples fixed in glutaraldehyde and osmicated before embedding. Contrasting with uranyl acetate and lead citrate was also performed after immunolabelling, but best results were obtained with uranyl acetate only. Glucagon-like immunoreactivity was located in round granules (300-600 nm) surrounded by a limiting membrane. The matrix varied in electron density and exhibited a dense core surrounded by a less dense mantle. The granules were seen in two different cell types, which differed in the electron density of their cytoplasm. Glucagon-immunoreactive cells were the largest pancreatic cells types and were often localized near somatostatin-containing cells.     

Simultaneous detection of multiple mutations conferring streptomycin resistance inMycobacterium tuberculosis using nanoscale engineered biomagnetites

Streptomycin-resistantMycobacterium tuberculosis has been attributed to two distinct classes of mutations, including point mutations within therpsL gene (three mutation sites) and therrs gene (seven mutation sites). We have developed an automated simultaneous detection system of multiple mutations based on thermal dissociation curve analysis for streptomycin resistance inM. tuberculosis using streptavidin-labeled bacterial magnetic particles (SA-BacMPs). With consideration for time and cost effectiveness, we used fewer PCR reactions, with a long PCR target (rpsL, 182 bp;rrs, 467 bp) including multiple mutation sites. In order to improve the amount of target DNA captured on BacMPs through streptavidin-biotin binding, several reaction conditions, such as salt species and concentration in the buffer, and reaction temperature were examined. Compared to the commonly used 1M NaCl solution, the amount of DNA captured on SA-BacMPs was about six times greater (approx 5 pmoles/50 μg BacMPs) in the 2M LiCl solution. Under these conditions, automated nucleotide discriminations of 10 targets inrpsL andrrs genes of streptomycin-resistant and wild-type strains were successfully performed at the same time.