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Journal of Integrative Agriculture 2023年第2期22卷 464-470页

作者： WANG Cui SUN Jin-jing YANG Xue-yong WAN Li ZHANG Zhong-hua ZHANG Hui-min College of Horticulture Qingdao Agricultural University/Engineering Laboratory of Genetic Improvement of Horticultural Crops of Shandong Province/Laboratory of Quality&Safety Risk Assessment for Fruit(Qingdao)Ministry of Agriculture and Rural AffairsQingdao 266109P.R.China Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of the Ministry of Agriculture and Rural Affairs/Sino-Dutch Joint Laboratory of Horticultural GenomicsBeijing 100081P.R.China

In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of *** most common ISH protocol uses paraffin wax;however,embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased ***,we developed an optimized protocol to simplify the process and improve RNA *** combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution,as in the whole-mount ISH method in the optimized *** the optimized protocol,we examined the expression patterns of the CLAVATA3(CLV3)and WUSCHEL(WUS)genes in shoot apical meristems and floral meristems of Cucumis sativus(cucumber)and Arabidopsis thaliana(Arabidopsis).The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin ***,the optimized protocol achieved high signal *** optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.

关键词： cucumber in situ hybridization Steedman’s wax paraffin wax

Detection of Human Papilloma Virus Type 16 E6 mRNA in Laryngeal Squamous Cell Carcinoma by in situ hybridization

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Chinese Journal of Cancer Research 2010年第3期22卷 218-223页

作者： Hai-rong Jiang Peng Wang Yong Li Tao Ning Xiao-song Rao Bao-guoLiu Department of Clinical Laboratory Chongqing Medical University Chongqing 630001 China Key Laborato＇ry of Carcinogenesis and Translational Research （M~inistry o~f＇Education） Department of Head and Neck Surgery Peking University School of OncologyBeijing Cancer Hospital ＆ Institute Beijing 100142 China Key Laboratory of Carcinogenesis and Translational Research （Ministry of Education） Department of Genetics Peking University School of Oncology Beijing CancerHospital ＆ Institute Beijing 100142 China Department of Pathology Peking University Shougang Hospital Beijing 100144 China

Objective：Laryngeal squamous cell carcinoma（LSCC） is a common malignant tumor in Northeast China and is frequently associated with well-established risk factors like smoking and alcohol *** papilloma virus（HPV） is an epitheliotropic oncogenic virus that has been detected in a variety of head and neck tumors including *** retrospective study was to investigate the prevalence of HPV infection in patients with ***：in situ hybridization was performed in 99 patients with LSCC to detect the expression of HPV-16 E6 ***：The positive rate of HPV16 E6 mRNA was 36.36%（36/99） in laryngeal squamous cell carcinoma（LSCC）,whereas only 3 of 50（6%） specimens of the normal laryngeal mucosa as a control group showed positive results（P0.05）.Additionally,there was no corelation between HPV16 and age,gender,clinical stage,nodal status and tumor site（P0.05）.Conclusion：The results suggest that the increased prevalence of HPV infection compared to normal laryngeal mucosa and the fact that high-risk HPV types（especially type 16） were the most frequently identified do not allow the exclusion of HPV as a risk factor in laryngeal squamous cell ***,their clinical value remains to be further investigated.

关键词： Laryngeal squamous cell carcinoma Human papilloma virus in situ hybridization

Reliable protocols for whole-mount fluorescent in situ hybridization （FISH） in the pea aphid Acyrthosiphon pisum： A comprehensive survey and analysis

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Insect Science 2014年第3期21卷 265-277页

作者： Chen-yo Chung Charles E. Cook Gee-way Lin Ting-Yu Huang Chun-che Chang Laboratory for Genetics and Development Department ofEntomologyllnstitute of Biotechnology College ofBioresources andAgriculture National Taiwan University Taipei Taiwan European Molecular Biology Laboratory European Bioinformatics Institute (EMBL-EBI) Wellcome Trust Genome Campus Hinxton Cambridge UK Research Center for Developmental Biology and Regenerative Medicine National Taiwan University Taipei Taiwan Institute of Biochemical Sciences College of Life Science National Taiwan University Taipei Taiwan Genome and Systems Biology Degree Program National Taiwan University Taipei Taiwan China

RNA in situ hybridization （ISH）, including chromogenic ISH （CISH） and fluorescent ISH （FISH）, has become a powerful tool for revealing the spatial distribution ofgene transcripts in model organisms. Previously, we developed a robust protocol for wholemount RNA CISH in the pea aphid Acyrthosiphon pisum, an emerging insect genomicmodel. In order to improve the resolving capacity of gene detection, we comprehensively surveyed current protocols of whole-mount RNA-FISH and developed protocols that allow,using confocal microscopy, clearer visualization of target messenger RNAs （mRNAs） - including those subcellularly localized and those with spatially overlapping expression. Wefind that Fast dye-based substrate fluorescence （SF）, tyramide signal amplification （TSA）, and TSA Plus all enable identifying gene expression thanks to multiplex amplificationof fluorescent signals. By contrast, methods of direct fluorescence （DF） do not allow visualizing signals. Detection of a single gene target was achieved with SF and TSA Plusfor most mRNAs, whereas TSA only allowed visualization of abundant transcripts such as Apvasl andAppiwi2 in the germ cells. For detection of multiple gene targets using doubleFISH, we recommend：（i） TSA/TSA, rather than TSA Plus/TSA Plus for colocalized mRNAs abundantly expressed in germ ceils, as proteinase K treatment can be omitted;and （ii） SF/TSA Plus for other gene targets such as Apenl and Apen2 as inactivation of enzyme conjugates is not required. SF/SF is not ideal for double FISH experiments due tosignal blurring. Based on these new conditions for RNA-FISH, we have obtained a better understanding of germline specification and embryonic segmentation in the pea *** anticipate that the RNA-FISH protocols for the pea aphid may also be used for other aphids and possibly other insect species, thus expanding the range of species from whichuseful insights into development and evolution may be obtained.

关键词： aphid fluorescence gene expression in situ hybridization model organism probe detection

P16 overexpression in pituitary adenomas studied by immunohistochemistry and in situ hybridization

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Chinese Medical Journal 2000年第2期113卷 66-70页

作者：易静于德蓉陈玉英熊文浩李骁雄沈建康汤雪明 Laboratory of Cell Biology Shanghai Second Medical University 280 Chongqing Road Shanghai 200025 China.

Objectives To examine the expression of p16 gene and the clinicopathological characteristics of 31 pituitary adenomas To investigate the relationship between p16 expression and cell proliferative activity by analyzing the expression of proliferating cell nuclear antigens (PCNA) Methods Immunohistochemistry and in situ hybridization were performed on freshly prepared paraffin embedded specimens of 31 pituitary adenomas Results Varying degrees of overexpression of the p16 gene was found in the tumors specimens A higher proportion of p16 positive tumor cells was associated with bigger size, more frequent invasion and higher recurrence of the tumors The expression of p16 was found to be significantly associated with the degree of PCNA expression Conclusion Though inactivation of the p16 gene is known in wide range of tumors, our study suggested that p16 overexpression might play a role in the development and progression of pituitary adenomas

关键词： p16 pituitary adenoma immunohistchemistry in situ hybridization

Detection of *** DNA in gastric epithelial cells by in situ hybridization

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World Journal of Gastroenterology 2002年第2期8卷 305-307页

作者： Xin-Liang Lu Ke-Da Oian Xun-Qiu Tang Yong-Liang Zhu Qin Du,Department of Digestive Diseases,Second Affiliated Hospital,Zhejiang University Medical College,Hangzhou 310009,Zhejiang Province,China Department of Digestive Diseases Second Affiliated Hospital Zhejiang University Medical College Hangzhou 310009 Zhejiang Province China. luxl@***

AIM: To investigate the presence of *** DNA within gastric epithelial cells in patients with *** infection and its possible carcinogenic mechanism. METHODS: Total 112 patients, with pathologically confirmed chronic superficial gastritis, chronic atrophic gastritis, intestinal metaplasia, atypical hyperplasia or gastric cancer were studied. Among them, 28 were *** negative and 84 *** positive. *** DNA in gastric epithelial cells was detected by GenPoint catalyzed signal amplification system for in situ hybridization. RESULTS: In the *** positive group, zero out of 24 chronic superficial gastritis (0.0%), four out of 25 precancerous changes (16.0%) and thirteen out of 35 gastric cancers (37.1%) showed *** DNA in the nucleus of gastric epithelial cells, the positive rates of *** DNA in the nucleus of gastric epithelial cells were progressively increased in chronic superficial gastritis, precancerous changes and gastric cancer groups (chi(2)=12.56, P=0.002); One out of 24 chronic superficial gastritis (4.2%), eleven out of 25 precancerous changes (44.0%) and thirteen out of 35 gastric cancers (37.1%) showed *** DNA in the cytoplasm of gastric epithelial cells (chi(2)=10.86, P=0.004). In the *** negative group, only one patient with gastric cancer was found *** DNA in the nucleus of gastric epithelial cells; Only two patients, one patient with precancerous changes and another with gastric cancer, showed *** DNA in the cytoplasm of gastric epithelial cells. Furthermore, *** DNA must have been in the cytoplasm as long as it existed in the nucleus of gastric epithelial cells. CONCLUSION: *** DNA exists both in the nucleus and the cytoplasm of gastric epithelial cells in patients with *** infections. The pathological progression from chronic superficial gastritis, precancerous changes to gastric cancer is associated with higher positive rates of *** DNA presence in the nucleus of gastric epitheli

关键词： in situ hybridization DNA, Bacterial Epithelial Cells Gastric Mucosa Helicobacter Infections Helicobacter pylori purification Humans Stomach Diseases

Analysis of hepcidin expression: in situ hybridization and quantitative polymerase chain reaction from paraffin sections

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World Journal of Gastroenterology 2012年第28期18卷 3727-3731页

作者： Yuhki Sakuraoka Tokihiko Sawada Takayuki Shiraki Kyunghwa Park Yuhichiro Sakurai Naohisa Tomosugi Keiichi Kubota Second Department of Surgery School of Medicine Dokkyo Medical University Kitakobayashi 880 Mibu Shimotsuga Tochigi 321-0293 Japan Proteomics Research Unit Division of Advanced Medicine Medical Research Institute Kanazawa Medical College Daigaku 1-1 Uchinada Kanazawa 920-0293 Japan

AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). METHODS: Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. RESULTS: Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. CONCLUSION: We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.

关键词： Hepcidin Expression in situ hybridization Immunohistochemistry Real-time polymerase chain reaction

Observation on in situ hybridization and Immunocytochemistry of Matrix Metalloproteinases in Rat Pancreas

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2003年第4期23卷 332-334页

作者：唐立华刘胜洪王芳刘子龙许耘王小丽李肇春 Department of Histology and Embryology Department of Reproductive Medicine

In situ hybridization and immunocytochemical techniques were employed to examine the expression of matrix metalloproteinases 1 (MMP 1) and to identify the pattern of its distribution in rat pancreas. The results indicated that the signal of MMP 1 mRNA and MMP 1 positive immunoreaction were detected in some fiberoblasts around interlobular ducts and exocrine cell in margin acinus of some lobules, but the signal of MMP 1 mRNA and MMP 1 positive immunoreaction could not be detected in most of other acinus and islets of pancreas. It is concluded that the expression of MMP 1 in above cells of rat might play an important role in acinar proliferation and differentiation of rat pancreatic tissues.

关键词： matrix metalloproteinases 1 in situ hybridization pancreas rat

SPECIFIC IDENTIFICATION OF HERPES SIMPLEX VIRUS IN HUMAN ESOPHAGUS WITH RAPID in situ hybridization IN 5 CASES

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Chinese Medical Sciences Journal 2008年第2期23卷 126-128页

作者： Ying-lan Gao Sung-sun Kim Chang-woo Han Yoo-duk Choi Jong-hee Nam Sang-woo Juhng Jun-shuo Jin Ling-fei Kong Chang-soo Park Department of Pathology Henan Provincial People＇s Hospital Zhengzhou 450003 Department of Pathology Chonnam National University Medical School Gwangju 501-746 South Korea Department of Surgery Inha University Medical School Incheon 400-712 South Korea

HUMAN herpes simplex virus esophagitis （HSVE） was first reported in 1940 by Johnson. ^1HSVE usually occurs in immunocompromised patients,such as those with acquired immunodeficiency syndrome （AIDS）, 2-4 malignancies, cutaneous burns, connective tissue diseases, inflammatory bowel disease, those taking immuno-suppressive therapy, and those undergoing organ transplantation,5 etc. In the immunocompetent individuals, HSVE is rare, having been reported in 39 cases and mainly affecting young males^6,7 The aim of this study was to delineate the clinical experience in the diagnosis of HSVE using rapid in situ hybridization and assess the various detection methods.

关键词： herpes simplex virus esophagus oligonucleotide DNA probe in situ hybridization

Localization of Avian Influenza Virus in Formalin-Fixed, Paraffin-Embedded Chicken Tissues by in situ hybridization

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Agricultural Sciences in China 2005年第12期4卷 931-936页

作者： ZHANG Wan-po GU Chang-qin BI Ding-ren SONG Nian-hua CHENG Guo-fu College of Veterinary Medicine Huazhong Agricultural University Wuhan 430070 PR.China Hubei Diagnosis Center of Animal Disease Wuhan 430064 P.R.China

In this study, in situ hybridization （ISH） was developed to detect avian influenza＇virus （AIV） in Madin-Darby canine kidney （MDCK） cells and formalin-fixed, paraffin-embedded chicken tissues. A cDNA probe corresponding to a region of AIV nucleoprotein （NP） gene was synthesized and labeled with digoxigenin. Probe specificity was determined by AIV infected MDCK cells in vitro and the results showed that strong cytoplasmic staining was only detected in AIV-infected cells. Various tissues were collected from 12 h to 35 days post-infection （PI） following inoculation with the H9N2 subtype A1V. AIV was localized in the epithelial cells of the duodenum and cartilage of the throat and trachea at 12 h PI. Tissues from uninfected chickens were negative. The finding of this study indicated ISH was a sensitive and specific technique to detect and localize AIV as well as to study AIV pathogenesis.

关键词： in situ hybridization Avian influenza virus cDNA probe Digoxigenin

in situ hybridization analysis of the expression of human telomerase reverse transcriptase (hTERT) in skin tumors

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In situ hybridization analysis of the expression of human te...