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1.
A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes acheived maximal sensitivity without use of biotin-streptavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin. 相似文献
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《Biotechnic & histochemistry》2013,88(4):228-232
With the introduction of microwave pretreatment, the quality of nonradioactive in situ hybridization (NISH) using DNA probes on formalin fixed tissue has significantly improved. Even after microwave treatment, however, there are cases where NISH results remain unsatisfactory. Therefore, we tried to improve NISH by testing other buffer systems as alternatives to the citrate buffer that is routinely applied during microwave pretreatment. By using buffer systems originally designed for immunohistochemistry, we significantly improved our NISH results. Difficult tissue samples were more accessible to NISH using these alternative buffer systems and made the quantitative evaluation easier. These results may also be of interest for combined applications of NISH and immunohistochemistry. 相似文献
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Chong-Hua Yao Sohei Kitazawa Takahiro Fujimori Sakan Maeda 《Biotechnic & histochemistry》1993,68(3):169-174
A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level. A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells. After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM). and others were embedded in Quetol for electron microscopy (EM). The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid. In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum. Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level. Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here. This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues. 相似文献
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Auday Maki Salwa Atwan Janan Al-Kaledar Andrew Beaman Robert Skoff 《Biotechnic & histochemistry》1997,72(1):38-44
Technical limitations are associated with conducting successful in situ hybridization. In this study, three cell types including a tumor neuroblastoma cell line (Neuro-2a), an oligodendrocyte primary culture, and a nonneuronal acute lymphoblastic leukemia cell line (Reh) were used to conduct successful nonradioactive in situ hybridization. Two cDNA probes were used. A 1 kb probe was used to identify the expression of proteolipid protein (PLP) mRNA in a primary culture of oligodendrocytes. A 760 bp cDNA was used to identify the expression of ubiquitin C-terminal hydrolase (UCH-L1) mRNA in Neuro-2a and Reh cells. The probes were labeled with digoxigenin-11-dUTP, denatured, and hybridized with cells fixed on coverslips. The efficiency of the labeling was tested using dot blot analysis by comparing the intensity of our labeled probes with known concentration of the probe labeled by the provider. The nonspecific signals were washed off, followed by detection of a signal specific to the gene. The specificity of the probes was determined by treating the cells with RNase A, hybridizing with bacterial Dig-labeled cDNA (pBR322) and hybridizing the tissues in the absence of labeled probe. During the labeling step, we found that addition of co-precipitants, such as tRNA or glycogen, during precipitation of the labeled probe followed by overnight incubation at -20 C is essential for good recovery of labeled cDNA. Dissolving the labeled probe in a buffer solution containing sodium dodecyl sulfate improves the quantity of the labeling. At the cellular level, prehybridization treatments optimize the permeability of the cell and allow efficient penetration of the labeled probe. Fixing with paraformaldehyde or an ethanol-acetic acid mixture can preserve the structure of cultured cells. To increase the signal to noise ratio, cells were treated with 0.2 N HC1 followed by extensive washes using a solution with a high salt concentration and containing dextran sulfate. This treatment significantly improves the signal and reduces the background in cell cultures, but not in tissue sections. The ability to reuse the labeled probe-hybridization mixture is another advantage for using nonradioactive in situ hybridization. 相似文献
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热启动PCR快速制备地高辛标记探针 总被引:7,自引:0,他引:7
介绍了一种在热启动PCR中,以Dig-11-dUTP部分代替dTTP,从少量基因组DNA中快速制备大量的地高辛标记的探针的方法,此探针灵敏度达0.03pg,并只和相关的DNA特异杂交. 相似文献
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The technique of in situ hybridization to both meiotic and mitotic chromosomes of Rumex acetosa is described. Differences in the efficiency of signal detection were observed between the two types of material. The implications of these results for in situ hybridization to other plant species are explored. 相似文献
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Akio Matsuhisa Yoshihiro Saito Yoshimasa Sakamoto Hiroyuki Keshi Hiroshi Ueyama Youko Aikawa Youichiro Kishi Tsuneya Ohno 《Microbiology and immunology》1994,38(7):511-517
We report herein the detection of intracellular bacteria in phagocyte-smears obtained from septicemia-suspected blood samples by in situ hybridization. This was obtained by using nick-translated biotin-11-dUTP-labeled DNA probes and streptavidin-alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia, 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast, only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria. Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus, our in situ hybridization method using the phagocyte-smears have diagnostic value for detecting most bacteria which cause septicemia. 相似文献
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We have investigated the applicability of human papillomavirus (HPV) DNA detection by in situ hybridization with biotinylated probes in epithelial cells obtained from the cervix using a cotton tip swab. We describe a simple procedure for obtaining homogeneous cell samples and good preservation of cellular structure. This is achieved by pretreatment of cells with L-cysteine before hybridization. Separate denaturation of cellular DNA and probe DNA is also necessary for satisfactory results. Both benign HPV DNA 6/11 and potentially oncogenic HPV DNA 16/18 could be identified in our series. In situ hybridization on cervical scrapes is a rapid, simple and very specific method for detecting patients infected with oncogenic HPV types. 相似文献
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两种非放射性标记方法在染色体原位杂交中敏感性的比较 总被引:1,自引:0,他引:1
通过原位杂交比较了地高辛配基和生物素标记探针,检测染色体单拷贝基因的敏感性。结果表明:在打点检测条上地高辛配基可检出30fg低限探针DNA,生物素为1pg。经原位杂交地高辛配基可检测出单拷贝基因,生物素未成功。 相似文献
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Molecular cytogenetics, particularly the localization of DNA sequences by in situ hybridization, has increased our understanding about the genomic structure of plants and animals. We demonstrate here the application of an improved nonfluorescent in situ hybridization system detection (DAKO GenPoint system) to plant chromosomes. Using this system, highly repetitive 18S-25S rRNA genes were mapped on Vicia faba chromosomes (2n = 12). The modified method of this horseradish peroxidase based enzymatic detection system gave satisfactory results that are comparable to fluorescent signal detection. 相似文献
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《Biotechnic & histochemistry》2013,88(1):6-22
We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes. 相似文献
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经1×10-6mol/L视黄酸诱导的P19细胞体外可向神经方向分化,接种于多聚赖氨酸(polyDlysine)和纤连蛋白(fibronectin)包被的玻片后,细胞逐渐聚集成团,此时细胞的贴壁性较差,进行原位分子杂交时容易脱落。我们尝试在细胞表面覆盖一层明胶,减少了细胞的脱落,又比较了蛋白酶K和胃蛋白酶对细胞蛋白质的消化作用,确定胃蛋白酶可较温和地消化细胞蛋白质,使探针有效地透入结合,杂交后细胞亦能较完整地保留于玻片上。 相似文献
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In Situ Hybridization of Lilium Whole Mount Synaptonemal Complex Chromosomal Preparations 总被引:1,自引:0,他引:1
Clare A. Hasenkampf 《Biotechnic & histochemistry》1991,66(4):210-215
Whole mount meiotic preparations of the synaptonemal complex complement of Lilium have been used for in situ hybridization experiments. A probe of the maize ribosomal DNA gene cluster has been successfully hybridized to the lily preparations. Three strong signals, corresponding to the three known lily nucleolus organizer regions, have been seen in most of the chromosome preparations. In situ hybridization experiments using meiotic preparations should be useful for identifying specific chromosomes, and for investigating the role of particular DNA molecules important to meiotic function. 相似文献
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Rapid Decalcification Using Microwaves for in Situ Hybridization in Skeletal Tissues 总被引:9,自引:0,他引:9
Motoharu Kaneko Tetsuya Tomita Takanobu Nakase Eiji Takeuchi Motoki Iwasaki Kazuomi Sugamoto Kazuo Yonenobu Takahiro Ochi 《Biotechnic & histochemistry》1999,74(1):49-54
In situ hybridization histochemistry is the sole tool available for detecting the localization and expression of specific RNA on histological sections under various in vivo conditions. For this paper, we examined the effect of microwave exposure on the time needed for decalcification of skeletal tissues and on the preservation of sensitivity for hybridization signals. Our data show that the use of microwave decalcification reduces the decalcification period while preserving intense hybridization signals for mouse al chain of procollagen type I mRNA in osteogenic cells in bone. The use of microwave treatment to decalcify skeletal tissues may prevent delay in obtaining experimental results or the loss of signals during in situ hybridization. 相似文献
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《Biotechnic & histochemistry》2013,88(2):54-78
Fluorescence in situ hybridization (FISH) is a powerful technique for detecting DNA or RNA sequences in cells, tissues and tumors. This molecular cytogenetic technique enables the localization of specific DNA sequences within interphase chromatin and metaphase chromosomes and the identification of both structural and numerical chromosome changes. FISH is quickly becoming one of the most extensively used cytochemical staining techniques owing to its sensitivity and versatility, and with the improvement of current technology and cost effectiveness, its use will surely continue to expand. Here we review the wide variety of current applications and future prospects of FISH technology. 相似文献