An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The 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 signals.Here,we developed an optimized protocol to simplify the process and improve RNA sensitivity.We 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 protocol.Using 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 wax.Moreover,the optimized protocol achieved high signal sensitivity.The optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.

In situ hybridization assay of androgen receptor gene in hepatocarcinogenesis

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Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modem lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the FI hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic （LA） hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that： at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell （PMC）, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva- lents were disjoined and most univalents were divided. Both the disjoined bivalents （half-bivalents） and the divided univalents （sister chromatids） moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the Fl LA hybrids and the variation of their BCx progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.

Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization

Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization （M-FISH） for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermediate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chromosomes in the whole genome of human tumors.

Dual fluorescence in situ hybridization in detection of HER-2 oncogene amplification in primary hepatocellular carcinoma

BACKGROUND: Molecular cytogenetics of oncogene HER-2 amplification in primary hepatocellular carcinoma (HCC) is still unknown. The aim of this study was to in vestigate the frequency of HER-2 oncogene amplification in primary HCC and its relations to clinicopathological pa rameters and prognosis. METHODS: Forty-two surgical samples from patients with primary HCC were detected for their HER-2 oncogene am plification. The number of chromosome 17 and their ratio were tested by dual fluorescence in situ hybridization (FISH) technique, and then the correlations between HER-2 amplification, clinicopathological characteristics and prog nosis were analyzed statistically. RESULTS: HER-2 oncogene amplification was detected in 9 (21.4%) of the 42 primary HCCs, including 4 patient with high copy (HC) (9.5%) and 5 patients with low copy (LC) (11.9%). HER-2 amplification was associated signifi cantly with tumor size and postoperative survival time o HCC patients (P<0.05), and the presence of HER-2 gene amplification was correlated with postoperative relapse (P— 0.257), but not related to sex, age, AFP level, HBV infec tion, histopathological grading and clinical staging of HCC patients (P>0.05). The HER-2 oncogene copy was exa mined in 31 (73.8%) of the 42 primary HCCs, consisting of 9 patients with HER-2 amplification (21.4%) and 22 pa tients with aneuploidy (52.4%). No significant relation were observed between the HER-2 oncogene copy, patien sex, tumor size, histopathological grading, clinical stag ing, postoperative relapse and survival time (P >0.05); bu the HER-2 oncogene copy was correlated significantly to age, AFP level and HBV infection (P <0.05). CONCLUSIONS: There are a lower frequency of HER-2 oncogene amplification and a higher frequency of chromo- some 17 aneuploidy in primary HCC. HER-2 oncogene amplification may be involved in the development and pro- gression of large HCC in some patients, and seems to be a valuably independent prognostic factor predicting the re- currence and poor survival in patients with large HCC.

Comparison of Fluorescence in situ Hybridization and Immunohistochemistry for Assessment of HER-2 Status in Breast Cancer Patients

The accurate assessment ofa proto-oncogene, human epidermal growth factor receptor-2 gene （HER-2）, is extremely important for the therapy and prognosis of breast cancer. Currently, immunohistochemistry （IHC） is the method widely used for the detection of HER-2 protein. Fluorescence in situ hybridization （FISH） has been suggested to be a golden standard assay for HER-2 amplification. This study examined the expression and amplification of HER-2 in paraffin-embedded sections of breast cancer tissues, and compared the two methods on the measurement of HER-2 status. HER-2 gene and protein were determined in breast cancer samples from 52 Chinese women by FISH and IHC respectively. The findings indicated that the HER-2 gene amplification was found in 18 cases （34.6%） by FISH and the HER-2 protein over-expression （score 3＋） in 15 cases （28.8%） by IHC. hnmunohistochemically, 28.6% of the cases scored as 2＋ and 93.3% of the cases scored as 3＋ were HER-2-positive by FISH. There was a significant correlation between the HER-2 gene amplification and HER-2 protein over-expression in breast cancer （P〈0.005）. No correlation was noted between the HER-2 gene amplification and any of the clinicopathological parameters examined, including age, menopausal status, menarche age, tumor size, histological tumor type, histological grade, lymph node status, and the expression of ER and PR. It was concluded that the detection of HER-2 gene amplification in breast cancer by FISH is valuable and can compare with HER-2 protein detection by IHC.

DETECTION OF WHITE SPOT SYNDROME VIRUS(WSSV) OF PENAEUS CHINENSIS BY IN SITU HYBRIDIZATION

White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I digested fragments of the WSSV genome were cloned; three of these fragments were used as non radioactive probes labeled with DIG 11 dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.

THE IMMUNOHISTOCHEMISTRY AND IN SITU cDNA-mRNA HYBRIDIZATION OF CARBAMYL PHOSPHATE SYNTHETASE I IN ENZYME-ALTERED LIVER CELLS DURING CARCINOGENESIS

The changes of carbamyl phosphate synthetase I(CPS 1)in diethylnitrosamine-(DEN)-inducedenzyme-altered liver cells were studied by means of immunohistochemical(PAP)and in situcDNA-mRNA hybridization methods.The experimental rats were treated with DEN,2-acetylaminofluorene(2-AAF)and 2/3 hepatectomy according to Solt-Farber’s protocol andwere further promoted by oral daily administration of 0.05% phenobarbital in drinking water.The results showed that the average number of lesions showing abnormal expression of CPS1 was relatively constant over the course of the experiment(8 months),while the numberof normally expressing lesions gradually decreased.The former lesions were also largerin volume than the latter ones.We conclude that in DEN-initiated lesions the abnormallyexpressed CPS 1 lesions may grow continuously,thus leading to the formation of largernodules.We also suspect that some of these lesions have increased tendencies to developinto tumors.

Development of taxonomic rRNA-targeted probes of two harmful algae: Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization

Harmful algal blooms recently have been under the spotlight throughout the world, because of their nega- tive impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were tak- en into consideration. The partial large subunit rDNA （D1-D2） of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization （FISH） tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K.. mikimotoi, respectively. Both the specific （taxonomic） （Pmin0443 and Kmik0602） and the control probes （UniC0512 and UniR0499） were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the tech- niques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant differ- ence （p 〉0.05） was observed in the cell densities of the samples determined by FISH and light microscopy （LM）. All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.

FLUORESCENCE IN SITU HYBRIDIZATION COMBINED WITH IMMUNOFLUORESCENT STAINING FOR RAPID DETECTION OF Nmyc AMPLIFICATION IN NEUROBLASTOMA

Objective: To establish a method to improve the detection of disseminated tumor cells in bone marrow and peripheral blood samples of neuroblastoma patients and analysis of cytogenetic aberration. Methods: Immunofluorescent staining was performed using a cocktail of primary monoclonal neuroblastoma antibodies (14.G2a, 5.1H11). Fluorescence in situ hybridization was applied with fluorescent probes specific for Nmyc genes afterwards. A novel computer assisted scanning system for automatic search, image analysis and repositioning of these positive cells was developed. Fifty-six bone marrow and peripheral blood samples from 7 patients were evaluated by this method. Results: Fluorescence in situ hybridization can be combined with immunofluorescent staining in detecting Nmyc amplification in neuroblastoma patients. Fluorescence in situ hybridization results correlated well with data obtained by conventional cytogenetic procedures. Conclusion: The technique described allows search of tumor cells in the bone marrow as well as detection of Nmyc amplification in interphase nuclei.

Observation on In Situ Hybridization and Immunocytochemistry of Matrix Metalloproteinases in Rat Pancreas

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.

Application of fluorescence in situ hybridization in the detection of bladder transitional-cell carcinoma: A multi-center clinical study based on Chinese population

Objective:To evaluate the diagnostic value of fluorescence in situ hybridization(FISH)in bladder cancer.Methods:We enrolled healthy volunteers and patients who were clinically suspected to have bladder cancer and conducted FISH tests and cytology examinations from August 2007 to December 2008.Receiver operating characteristic(ROC)curve analysis was performed and the area under curve(AUC)values were calculated for both the FISH and urine cytology tests.Results:A cohort of 988 healthy volunteers was enrolled to establish a reference range for the normal population.A total of 4807 patients with hematuria were prospectively,randomly enrolled for the simultaneous analysis of urine cytology,FISH testing,and a final diagnosis as determined by the pathologic findings of a biopsy or a surgically-excised specimen.Overall,the sensitivity of FISH in detecting transitional-cell carcinoma was 82.7%,while that of cytology was 33.4%(p<0.001).The sensitivity values of FISH for non-muscle invasive and muscle invasive bladder transitional-cell carcinoma were 81.7%and 89.6%,respectively(p=0.004).The sensitivity values of FISH for low and high grade bladder cancer were 82.6%and 90.1%,respectively(p=0.002).Conclusion:FISH is significantly more sensitive than voided urine cytology for detecting bladder cancer in patients evaluated for gross hematuria at all cancer grades and stages.Higher sensitivity using FISH was obtained in high grade and muscle invasive tumors.

Optimization and application of fluorescent in situ hybridization (FISH) process in EBPR fed with municipal wastewater

For sludge samples from EBPR reactor fed with municipal wastewater,fluorescent in situ hybridization (FISH) operation process including moisture chamber,pretreatment,treatment with lysozyme and Proteinase K and washing time was optimized and improved.Preserving box was chosen to be moisture chamber due to its bigger depth /radii ratio,good sealability and big volume contrast with Petri dish.3-5 mm diameter glass balls could disperse samples without destroying microorganism cells and community structure.Impurities and ECPs could be removed easily and sludge samples became thinner after dispersing which benefit the observation.Permeabilized cells with lysozyme and Proteinase K could enhance probe penetration before hybridization.Experiments of different treatment time with lysozyme and Proteinase K were carried out.Best results were observed when sludge samples treated with lysozyme 10 min/Proteinase K 20 min or lysozyme 20 min/Proteinase K 10 min.Slides were washed at 48 ℃ for 10,20,30,40 and 60 min in parallel.The best washing time was 20 min when washing temperature was 48 ℃.Fluorescent dye could residue when washing time was 10 min and washing out happened when washed for 30 min or more.

DETECTING EXPRESSION OF MRP-1/CD9 mRNA IN LUNG CANCERS USING TISSUE MICROARRAYS AND FLUORESCENCE IN SITU HYBRIDIZATION METHODS

Objective： The aim of this study was to investigate the MRP-1/CD9mRNA expression in lung cancer and normal lung tissues and the relationship between its expression and pathologic grades, clinical stages, metastasis and prognosis. Methods： To observe MRP-1/C9mRNA expression, tissue microarray （TMA） containing 54 lung cancers and 10 normal lung tissues was prepared and Fluorescence in situ hybridization was used. Results： The positive rate of MRP-1/CD9 expression was 48.1% in lung cancer, lower than that of normal lung tissues. The statistical difference was significant （P〈0.05）. Its protein expression had no relationship with the patients＇ ages, sex and the macroscopic type of tumor, but had relationships with the histological type, clinical stage, differentiated degree and metastasis. The expression in non-small cell lung cancer （NSCLC） was higher than that in small cell lung cancer （SCLC）; in well-moderately differentiated group was higher than that in poorly differentiated group; Earlier period group （I＋II） was higher than in later period group （Ⅲ＋Ⅳ）; and in group without lymphoid metastasis was higher than in patients with lymphoid metastasis. Conclusion： The progression of the lung cancer maybe related with the descended MRP-1/Cd9 expression, which may be useful in evaluating the prognosis of cancer patients.

Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy

H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.

Distribution characteristics of ammonia-oxidizing bacteria in the Typha latifolia constructed wetlands using fluorescent in situ hybridization(FISH)

A molecular biology method, fluorescent in situ hybridization（FISH）, in which the pre-treatment was improved in allusion to the media of the constructed wetlands（CW）, e.g. the soil and the grit, was used to investigate the vertical distribution characteristics of ammonia-oxidizing bacteria（AOB） quantity and the relation with oxidation-reduction potential（ORP） in the Typha latifolia constructed wetlands under three different Ioadings in summer from May to September. Results showed that the quantity of the AOB decreased in the Typha latifolia CW with the increase of vertical depth. However, the AOB quantity was 2-4 times the quantity of the control in the root area. Additionally, ORP in the rhizosphere was found to be higher than other areas, which showed that Typha latifolia CW was in an aerobic state in summer when using simulated non-point sewage at the rural area of Taihu Lake in China and small town combined sewage.

Literature Analysis on Fluorescence in situ Hybridization in China during 2002-2016

In order to explore researches about the chromosome karyotype analysis and fluorescence in situ hybridization(FISH) technology in China,using the bibliometric method,taking " fluorescence in situ hybridization(FISH) " and " chromosome" as key words,this paper made a statistical analysis on the literature published in China National Knowledge Infrastructure(CNKI) during 2002-2016.The results indicated that the number of papers published in 2002 was the smallest(37),while the number of papers published in 2012 was the largest(125).In terms of the distribution of organizations of authors,in 1201 papers,11 organizations published papers ≥15,accounting for 21.65%.In terms of distribution of papers published by different periodicals,11 periodicals published papers ≥10,accounting for 17.65%.In terms of the papers supported by foundation projects,in all papers searched,377 papers were supported by foundation projects,accounting for 31.39%.In terms of the distribution of doctoral and master's dissertations,259 papers were master's dissertations,accounting for 21.57%;92 papers were doctoral dissertations,accounting for 7.66%.

Niche Differentiation of Phenol-Degrading Microorganisms in UASB Granular Sludge as Revealed by Fluorescence in situ Hybridization

A microbial community structure of granules harvested from an anaerobic sludge blanket reactor treating phenolic wastewater was investigated using fluorescence in situ hybridization(FISH)and clone library construction.Clones of Syntrophorhabdaceae and Cryptanaerobacter were observed to be responsible for phenol degradation.For accurate taxonomic assignment of Cryptanaerobacter clones,phylogenetic analysis using nearly full-length 16S ribosomal RNA(rRNA)gene sequences was necessary.Three oligonucleotide probes were designed to detect the following three taxonomic groups:Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus.FISH analysis of thin sections of anaerobic granules showed a random distribution of bacteria and archaea.However,a well-defined distribution of Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus was observed.Cryptanaerobacter and Syntrophus were found on the outer layer of the granules and were closely associated with each other,while Syntrophorhabdaceae was located in the deeper part of the granules.Such specific distribution of the bacteria is most likely due to their metabolic association and affinity for the substrate.Phenol degradation in the granular sludge was observed to be carried out in the following way.First,Cryptanaerobacter converts phenol to benzoate,which is then degraded by Syntrophus into acetate.This syntrophic degradation of phenol occurs near the surface of the granule,where the phenol concen-tration is high.In the deeper part of the granule,where the phenol concentration is lower,Syntrophorhabdaceae degrades phenol into acetate.We observed that Syntrophorhabdaceae is less likely to produce benzoate as an intermediate to feed the neighboring organisms,which contradicts the theo-ries presented by previous studies.

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

Oryza sativa (一个染色体) 和 O 的 genomic 结构。meyeriana (G 染色体) 比较地在 situ 杂交(GISH ) 用二色的 genomic 被学习。GISH 清楚地能在 O 的染色体之间区别。sativa 和 O。在没有堵住 DNA 的种间的 F1 混血儿的 meyeriana，和合作杂交几乎没被检测。O 的平均有丝分裂的染色体长度。meyeriana 被发现是乘 O 的的 1.69。sativa。染色的 4,6-diamidino-2-phenylindole 的比较出现了 O 的染色体。meyeriana 更广泛地被标记，建议 G 染色体与更重复的序列比被放大一个染色体。在分裂期间原子核， 9-12 多彩石印版中心通常被检测，将近，所有多彩石印版中心组成了 G 染色体特定的 DNA。中心由相应于 G 染色体的染色质压缩形成了的更多和更大的多彩石印版与它的父母相比在混血儿被检测。在 F1 混血儿的 pachytene 期间， A 和 G 的大多数染色体互相，除了 1-2，染色体在他们的手臂的结束配对的触处。在 meiotic 中期我， chromosomal 协会的三种类型，即 O。sativa-O。sativa (A-A ) ， O。sativa-O。meyeriana (A-G ) 和 O。meyeriana-O。meyeriana (G-G ) ，在 F1 混血儿被观察。配对配置的 A-G 染色体包括了 bivalents 和 trivalents。结果向学习染色体组织和 O 的进化提供了一个基础。meyeriana。