Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labelled probes

Summary Conditions suitable for immunogold detection of digoxigenin-labelled DNA probes hybridized to parvovirus B19-infected erythroid cells embedded in Lowicryl K4M and LR White acrylic resins were established at the electron microscope level. The protocol was initially optimized using a positive control probe for whole human DNA which produced signal over the heterochromatin of all nucleated cells. In cultures harvested 2 days postinfection, B19 nucleic acid was detected mainly within the centrinuclear region of erythroid cells exhibiting characteristic margination of the chromatin. The B19 hybridization signal was largely unaffected by denaturation and was resistant to RNase digestion but sensitive to DNase digestion, indicating that it was mainly single-stranded B19 DNA. Relatively few gold particles were found over crystalline arrays of viral capsids, consistent with the observation that they are composed of mainly empty capsids. B19 nucleic acid was detected in apparent transit from nucleus to cytoplasm through pores in the nuclear membrane. While the sensitivity of this system is limited by the fact that hybridization occurs only at the surface of the section, it is a rapid and specific means of localizing viral nucleic acids with a high degree of resolution.     

In situ localization of enzymes and mucin in normal rat colon embedded in plastic

Summary Several enzymes were investigated histochemically in the colons of normal male F344 rats in order to understand the function of different types of cells in this tissue. Serial methacrylate-embedded sections (2–4 µm) allowed the precise localizations of several enzymes including acid phosphatase, alkaline phosphatase, -glutamyl transpeptidase,N-acetyl--d-glucosaminidase (hexosaminidase), -naphthyl butyrate esterase and 5-nucleotidase. Sites reactive with periodic acid-Schiff were also localized. Gradients of enzyme activity were observed between caecum and rectum and/or from the luminal surfaces to the bases of the crypts for hexosaminidase, esterase and -glutamyl transpeptidase. To our knowledge this is the first histochemical demonstration of -glutamyl transpeptidase in normal rat colonic epithelial cells. The utilization of the methacrylate-embedding technique has revealed previously undescribed gradients of enzyme activity and has allowed the localization of enzyme activities not previously reported in normal rat colonic mucosa.     

Analysis of cell ploidy in histological sections of mouse tissues by DNA-DNA in situ hybridization with digoxigenin-labelled probes

Summary DNA-DNA in situ hybridization, with two digoxigenin-labelled, chromosome-specific DNA probes, was used to determine the number of copies of a given chromosome in interphase nuclei and so identify putatively polyploid nuclei in histological sections of several mouse tissues. One hybridization site per diploid genome was expected for tissues with hemizygous markers: male mice hybridized with a Y chromosome probe (pY353/B) or hemizygous transgenic mice hybridized with a -globin probe (pM02). Nuclei with more than one hybridization site were considered putative polyploids. Three groups of experiments were undertaken: (1) evaluation of the method, using mouse liver sections; (2) studies of tissues already known to contain polyploid nuclei, and (3) studies that resulted in the discovery that the mouse ovary contains polyploid nuclei. First, control studies showed that the ability to detect the target DNA sequences was affected by section thickness. Studies of nuclear ploidy in the developing mouse liver revealed a pattern similar to that established by previous studies using DNA content as a criterion for ploidy. At birth, only about 5% of the liver nuclei were polyploid; this increased to 10–15% by 10–20 days and was followed by a sharp increase in the frequency of tetraploid nuclei between 20 and 40 days (to about 35%) and a more gradual increase in higher order polyploid nuclei. Secondly, this technique was used to confirm that polyploid (mostly tetraploid) nuclei were present in the bladder epithelium, heart, uterine decidua and placental trophoblast. Higher order polyploidy was seen in large bone marrow cells (megakaryocytes) but not in the even larger trophoblast giant cells of the placenta, thus confirming previous claims that these cells are polytene rather than polyploid. Thirdly, putatively tetraploid nuclei were found in the ovarian follicle and corpus luteum. As far as we are aware, this is the first time polyploid nuclei have been reported for the mouse ovary.     

The detection of the mRNAs of procollagen types I, II and III in human fetal fingers by in situ hybridization using digoxigenin-labelled oligonucleotide probes

Summary Messenger RNAs (mRNAs) encoding procollagen 1 type I, 1 type II and 1 type III have been localized in paraffin sections of human fetal fingers using digoxigenin-labelled synthetic oligonucleotide probes. The probe-mRNA hybrids were visualized using an anti-digoxin antibody amplified with sandwich techniques. These protocols provided an excellent hybridization signal with minimal background noise. The sensitivity of the protocols was nearly equivalent to that seen when using isotopic cDNA probes. In human fetal fingers, intense hybridization signals for procollagen 1 type I mRNA were detected in the osteoblasts and the fibroblasts of periosteum and perichondrium, the tenocytes of tendons, fibroblasts of ligaments, the synovial membrane and deeper layers of the dermis. In contrast, positive hybridization signals for procollagen 1 type II mRNA were visualized in chondrocytes and the cambial layer of perichondrium. The signals for procollagen 1 type III mRNA were detected in the fibroblasts of the dermis and perichondrium. The probes which have lower melting temperatures (Tm) could not detect the corresponding mRNAs.     

In situ hybridization comes of age

Dr Coulton is a lecturer in the Department of Biochemistry at Charing Cross and Westminster Medical School, London W6 8RF. His principal research interest is in the molecular and cellular basis of inherited disease of skeletal muscle.     

In situ hybridization: Alkaline phosphatase visualization of biotinylated probes in cryostat and paraffin sections

Summary Alkaline phosphatase immunochemical systems were evaluated for use in the demonstration ofin situ hybridized biotin-labelled probes in frozen and fixed sections of tonsil. Three probes were used: total genomic DNA, pHY2.1, a human repetitive sequence which hybridizes to a 2.12 KB sequence on the Y chromosome (2000 repeats) and a 2.0 KB sequence on the autosomes (100–200 repeats), and human papilloma virus type II. Indirect, three- and five-stage detection methods were compared on cryostat sections. The indirect method involved the application of a streptavidin, biotinylated alkaline phosphatase sequence. The three-stage procedure comprised a mouse monoclonal anti-biotin, rabbit anti-(mouse immunoglobulin), mouse APAAP system. In the five-stage method the indirect and three-stage reagents were sequentially applied. Alkaline phosphatase was demonstrated using a Fast Red naphthol-capture method.The total genomic DNA probe was used initially to investigate hybridization conditions including the optimum temperature of denaturation, which was found to be higher than previously reported. The five-stage detection method gave the most sensitive results for the Y sequence probe, with intense demonstration of the Y body in male nuclei and autosomal sequences in female nuclei. This method was then applied to fixed tissue sections and gave Y body signals on Bouin's and Carnoy's fixed tissue. On the other hand tissue fixed using formalin-based solutions required proteolytic digestion as a pretreatment to hybridization for a Y body signal. The application of this methodology to viral diagnosis in routine fixed anogenital tissue and cytological preparations was also demonstrated.     

In situ measurements of enzyme activities in the brain

Summary The present review focusses on enzymes involved in the metabolism of amino acid neurotransmitters and the microphotometric determinations of their activities in various layers of the rat hippocampus. The enzymes are NAD-linked isocitrate dehydrogenase (NAD-ICDH), glutamate dehydrogenase (GDH), and GABA transaminase (GABAT), all of which are localized in mitochondria. GDH seems to be restricted to astrocytes, whereas NAD-ICDH and GABAT are localized in neurons as well as in astrocytes. NAD-ICDH is an important enzyme of the tricarboxylic acid cycle and may deliver -ketoglutarate for the formation of glutamate and GABA, which serve as neurotransmitters in the hippocampus. GDH catalyses the interconversion of -ketoglutarate and glutamate, whereas GABAT is the important GABA-degrading enzyme and requires -ketoglutarate for its activity. While differing in their cellular distribution and activity levels, NAD-ICDH, GDH and GABAT are significantly correlated in their hippocampal distribution. Furthermore, developmental and pharmacohistochemical studies suggest that the distribution and activity of astrocytic GDH is correlated with amino-acidergic neurotransmission in the hippocampus. The data reported give further evidence for a metabolic relationship between neurons and astrocytes in the turnover and metabolism of glutamate and GABA.     

巴西橡胶树HbSUT3和HbSUT5 mRNA在嫩茎和中脉中的原位杂交分析

为研究巴西橡胶树(Hevea brasiliensis)中HbSUT3和HbSUT5基因的功能,采用地高辛标记的RNA探针与橡胶树嫩茎和中脉两种组织切片分别进行RNA原位杂交,对这2种SUT基因在组织中的表达区域与表达特点进行了分析。结果表明,在橡胶树嫩茎中,两个SUT基因主要在树皮的韧皮部和皮层细胞中表达;在中脉中,两个SUT基因在除木质部导管系统外的其它部位均有表达;HbSUT3基因在嫩茎和中脉中的表达量相近,而HbSUT5基因在嫩茎中的表达量远高于中脉。这些揭示HbSUT3和HbSUT5基因可能广泛参与韧皮部装载、蔗糖运输与库细胞供给等活动,同时两个SUT基因也存在功能分化。     

棉花细菌人工染色体的荧光原位杂交(BAC-FISH)技术

细菌人工染色体荧光原位杂交(BAC-FISH)技术是植物染色体识别、物理作图等分子细胞遗传学研究的重要工具,但对于某些物种尤其是多倍体植物,由于大量重复序列的存在等问题,使得该技术应用受到很大的限制.通过选择棉花分子遗传图中高重组区的微卫星位点(simple sequence repeats,SSR)标记的策略,筛选到不含或含有少量重复序列的细菌人工染色体(BAC)克隆,同时,在通用FISH技术程序基础上,通过改进发根、变性、洗脱条件等步骤,构建出适合于棉花的BAC-FISH技术,简化了操作流程的同时,获得稳定的杂交结果及较高的检出率;并通过将一随机获得的BAC进行染色体的物理定位,进一步引入双探针、双色及重复杂交技术,显示了该技术的成熟与良好的应用前景和价值.     

In situ labelling of vascular endothelium with fluorescent acetylated low density lipoprotein

Summary Acetylated low density lipoprotein is metabolized by a receptor-mediated process in endothelial cells. We have used the lipoprotein labelled with the fluorescent probe 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate to localize endothelial cells lining blood vessels. Following intravenous injection of the labelled lipoprotein, the vascular sinusoids and all other hepatic blood vessels were clearly labelled in cryostat sections of mouse liver. The endothelium of other organs such as brain, kidney, and testis was also brightly labelled. In addition, the lipoprotein was used to label the endothelium of bovine aorta, the vasculature in the chick chorioallantoic membrane and the vessels in a growing murine melanoma. These results demonstrate that the fluorescent labelled lipoprotein can be used forin situ labelling of the endothelium from large as well as small blood vessels in a variety of species.     

In situ heterogeneity of peroxisomal oxidase activities: An update

Summary Oxidases are a widespread group of enzymes. They are present in numerous organisms and organs and in various tissues, cells, and subcellular compartments, such as mitochondria. An important source of oxidases, which is investigated and discussed in this study, are the (micro)peroxisomes. Oxidases share the ability to reduce molecular oxygen during oxidation of their substrate, yielding an oxidized product and hydrogen peroxide. Besides the hydrogen peroxide-catabolizing enzyme catalase, peroxisomes contain one or more hydrogen peroxide-generating oxidases, which participate in different metabolic pathways. During the last four decades, various methods have been developed and elaborated for the histochemical localization of the activities of these oxidases. These methods are based either on the reduction of soluble electron acceptors by oxidase activity or on the capture of hydrogen peroxide. Both methods yield a coloured and/or electron dense precipitate. The most reliable technique in peroxisomal oxidase histochemistry is the cerium salt capture method. This method is based on the direct capture of hydrogen peroxide by cerium ions to form a fine crystalline, insoluble, electron dense reaction product, cerium perhydroxide, which can be visualized for light microscopy with diaminobenzidine. With the use of this technique, it became clear that oxidase activities not only vary between different organisms, organs, and tissues, but that heterogeneity also exists between different cells and within cells, i.e. between individual peroxisomes. A literature review, and recent studies performed in our laboratory, show that peroxisomes are highly differentiated organelles with respect to the presence of active enzymes. This study gives an overview of thein situ distribution and heterogeneity of peroxisomal enzyme activities as detected by histochemical assays of the activities of catalase, and the peroxisomal oxidasesd-amino acid oxidase,l--hydroxy acid oxidase, polyamine oxidase and uric acid oxidase.     

In situ hybridization technique using an immunogold silver staining system

Summary Anin situ hybridization technique using an immunogold silver staining detection system was used to detect biotinylated DNA probes in cultured cells and in formalin-fixed paraffin-embedded tissue. The detection method is rapid, reliable and economical, producing an insoluble signal at the site of hybridization which is visible at low-power light microscopy and which can be enhanced by epipolarization microscopy.     

无细胞系统原位制备蛋白质芯片及其应用

主要介绍了目前在生物芯片表面进行蛋白质无细胞表达与定向制备蛋白质芯片的研究进展,包括各种基因植入芯片的方法、蛋白质体外不同表达的途径、蛋白质固定的策略以及可能的应用发展前景等．蛋白质芯片以其高通量、高灵敏和检测迅速等优点正成为蛋白质组学研究中的重要工具之一．蛋白质的高效表达与纯化、蛋白质在芯片表面的有效固定与蛋白质活性的保持等内容是蛋白质芯片技术发展的关键．采用纳米生物技术与无细胞表达系统,已经可以在生物芯片表面通过植入基因的方式制备相关的蛋白质芯片,从而为蛋白质芯片的原位制备开辟了新的方向．     

植物酶的组织和细胞化学定位研究方法

酶是参与植物体内生化反应的特殊蛋白质。在保持活组织和细胞结构完整性的条件下,利用组织化学、细胞化学、免疫学和显微检测等技术研究酶的即位定位,是了解酶在组织、细胞和亚细胞中的分布、活性动态与定量及酶功能等的重要途径。对植物体中酶定位的组织化学和细胞化学方法的概念、原理与研究进展进行了综述,并根据国际酶化学分类编号顺序,分别介绍了25种酶的组织化学染色定位所用的反应介质和染色方法及46种酶的细胞化学定位方法的参考文献。     

In situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels

Ornithine transcarbamylase deficiency is a human genetic disease potentially susceptible to gene therapy. A murine model system exists for the disease in the sparse-fur (spf) mouse. Before gene therapy studies can be performed it is necessary to have practical methods which could detect successful gene transfer. Therefore we have developed an in situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels. Following electrophoretic separation under nondenaturing conditions inorganic phosphate cleaved from carbamyl phosphate in gels as a result of enzymatic activity was precipitated as phosphomolybdic acid and visualized by reduction with ascorbic acid. Results from the procedure correlated with ornithine transcarbamylase activity as measured by solution assay for citrulline, the other product of the reaction. This procedure readily distinguished mutant forms of ornithine transcarbamylase as exemplified by the murine spf mutation and resolved ornithine transcarbamylases of all animals tested into multiple forms. The procedure further distinguished ornithine transcarbamylases of animals of several different genera while yielding virtually identical patterns of the enzyme from species within the same genus. This procedure also suggested that the human enzyme was more labile than murine ornithine transcarbamylase; direct thermolability studies confirmed this finding.