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Mapping Neurotransmitter Identity in the Whole-Mount <i>Drosophila</i> Brain Using Multiplex High-Throughput Fluorescence <i>in Situ</i> Hybridization
摘要： Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence in situ hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount Drosophila melanogaster brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in Drosophila brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter co-expression, reveal neurotransmitter phenotypes of specific cell types and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large scale FISH detection of any mRNA in whole-mount fly brains.
关键词： Neurotransmitter,mRNA,Fluorescence in situ hybridization,Gene expression,Drosophila
更新于2025-11-21 11:08:12
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Graphene oxide-quenching-based fluorescence in situ hybridization (G-FISH) to detect RNA in tissue: Simple and fast tissue RNA diagnostics
摘要： FISH-based RNA detection in paraffin-embedded tissue can be challenging, with complicated procedures producing uncertain results and poor image quality. Here, we developed a robust RNA detection method based on graphene oxide (GO) quenching and recovery of fluorescence in situ hybridization (G-FISH) in formalin-fixed paraffin-embedded (FFPE) tissues. Using a fluorophore-labeled peptide nucleic acid (PNA) attached to GO, the endogenous long noncoding RNA BC1, the constitutive protein β-actin mRNA, and miR-124a and miR-21 could be detected in the cytoplasm of a normal mouse brain, primary cultured hippocampal neurons, an Alzheimer’s disease model mouse brain, and glioblastoma multiforme tumor tissues, respectively. Coding and non-coding RNAs, either long or short, could be detected in deparaffinized FFPE or frozen tissues, as well as in clear lipid-exchanged anatomically rigid imaging/immunostaining-compatible tissue hydrogel (CLARITY)-transparent brain tissues. The fluorescence recovered by G-FISH correlated highly with the amount of miR-21, as measured by quantitative real time RT-PCR. We propose G-FISH as a simple, fast, inexpensive, and sensitive method for RNA detection, with a very low background, which could be applied to a variety of research or diagnostic purposes.
关键词： glioblastoma multiforme tumor,tissue RNA diagnostics,Graphene oxide-quenching-based fluorescence in situ hybridization (G-FISH),Alzheimer’s disease,formalin-fixed paraffin-embedded (FFPE) tissue
更新于2025-09-23 15:23:52
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Preparation of fluorescent in situ hybridisation probes without the need for optimisation of fragmentation
摘要： DNA-fluorescence in situ hybridisation (DNA-FISH) allows visualisation of chromosome organisation and fluorescently labelled DNA fragments that are often produced from rearrangement. FISH probes are pools of short template plasmids that contain large genomic inserts. For effective sample penetration and target hybridisation it is critical that probe fragments are between 200 and 500bp. Production of these short probes requires significant optimisation and can be confounded access to expensive sonication equipment or inherent sequence features that influence enzymatic fragmentation or amplification. Here we demonstrate that effective FISH probes can be prepared without the need for optimisation of fragmentation using a cocktail of two the 4bp recognition sequence restriction enzymes CviQI and AluI.
关键词： Cancer,Fluorescence in situ hybridization,Translocation,FISH,Probe
更新于2025-09-23 15:23:52
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Peptide Nucleic Acid–Fluorescence In Situ Hybridization for Detection of Staphylococci From Endophthalmitis Isolates: A Proof-of-Concept Study
摘要： PURPOSE. Rapid identi?cation of pathogens causing endophthalmitis may improve treatment outcomes through early administration of species-speci?c medication. The current study reports a new molecular application of peptide nucleic acid–?uorescence in situ hybridization (PNA-FISH) with Staphylococcus-speci?c molecular PNA probes for the potential rapid detection of common pathogens causing endophthalmitis. METHODS. An experimental study was designed to evaluate the proof of concept at the microbiology laboratory of the Bascom Palmer Eye Institute. Stored culture-positive staphylococci endophthalmitis isolates obtained from prior vitreous samples (n ? 15), along with broth as negative controls (n ? 5) were used. Inoculum was prepared to a ?nal concentration of 1 3 105 colony-forming units/mL to ensure that the isolates were viable. Smears of samples were ?xed and hybridized using QuickFISH protocol with probes for Staphylococcus. RESULTS. With PNA-FISH technique, Staphylococcus aureus was identi?ed in 9 of 10 samples and coagulase-negative staphylococci were identi?ed in 10 of 10 samples. Detection time was 20 minutes. CONCLUSIONS. This study serves a proof of concept using a new microbial detection system with FISH probes, and may have the potential for clinical use in the rapid and accurate identi?cation of isolates from patients with endophthalmitis.
关键词： peptide nucleic acid (PNA),endophthalmitis,rapid identification,fluorescence in situ hybridization (FISH)
更新于2025-09-23 15:22:29
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Fluorescence in situ hybridization in 1?mL of selective urine for the detection of upper tract urothelial carcinoma: a feasibility study
摘要： Kidney-sparing surgery of upper tract urothelial carcinoma (UTUC) requires a stringent follow-up with frequent ureteroscopies. Triage testing could reduce the number of follow-up ureteroscopies and hence minimize the invasiveness of follow-up. The use of urine-based markers for triage seems appealing but should be feasible with selective urine from outpatient cystoscopy to maximize the reduction of invasiveness. In this study, the feasibility of UroVysion? fluorescence in?situ hybridization (FISH) for the detection of UTUC in 1?mL of selective urine is investigated. Ten consecutive patients with biopsy-proven UTUC and five patients with negative diagnostic ureteroscopy findings were included in this case-control study. During ureteroscopy, 1?mL of selective urine was collected passively with a ureteral splint for Urovysion? FISH. The FISH rater was blinded to any clinical information. The results of FISH were compared to the findings of concomitantly collected selective urine cytology and the patients’ UTUC status. FISH was feasible in all samples with a sensitivity of 90% and a specificity of 80% for UTUC. In comparison, selective cytology resulted in a diagnostic yield of 87% with a sensitivity of 80% and a specificity of 67%. In conclusion,?UTUC detection is feasible with FISH in 1?mL of passively collected selective urine. Thus from a technical point of view, FISH could be used as an outpatient triage test to decide if follow-up ureteroscopy is necessary after kidney-sparing surgery of UTUC. Evaluation of the diagnostic accuracy of FISH for the suggested pathway deserves further attention.
关键词： Ureteroscopy,Urothelial carcinoma,Cystoscopy,Fluorescence in?situ hybridization,Urine cytology,Upper tract urothelial carcinoma
更新于2025-09-23 15:21:21
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Fluorescence in situ hybridization (FISH): History, limitations and what to expect from micro-scale FISH?
摘要： In this article, we review an important cytogenetic technique - fluorescence in situ hybridization (FISH) - which is used for obtaining spatial genomic and transcriptomic information. FISH is widely utilized in genomic and cell biological research as well as for diagnostic applications in preventive and reproductive medicine, and oncology. It is the gold standard technique for the detection of chromosomal abnormalities. Despite the high specificity of FISH and the possibility of direct quantitative imaging, some of its key limitations prevent its regular use in diagnostics. To promote the extensive use of FISH for these applications, limitations in assay time and probe consumption will need to be addressed. Microfluidic technologies hold great promise in improving exactly these parameters. In the past two decades, microtechnology has matured and enabled a new line of analysis tools for biomedical and chemical sciences. Incidentally, the convergence of microtechnology with microfluidics is starting to have a decisive impact in the field of medical diagnostics. By miniaturizing implementations of diagnostic assays, the special characteristics of fluid flow in small volumes can be leveraged to modify reaction kinetics and thus reagent delivery time of assays. Here we highlight selected important historical views on FISH, review its current implementations, and provide a perspective on the future developments and the micro-scale implementations of FISH.
关键词： Diagnostics,Hybridization kinetics,Microfluidics,Tissue sections,Fluorescence in situ hybridization
更新于2025-09-23 15:21:01
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Synovial Sarcoma Diagnosis on Fine Needle Cytology Sample Confirmed by Fluorescence in Situ Hybridization
摘要： Synovial sarcoma (SS) is a mesenchymal neoplasm usually affecting adolescents and young adults, with one third of cases occuring in the first two decades of life. SS is characterized by the specific balanced chromosomal translocation t(X;18)(p11;q11), resulting in the fusion of SYT gene on chromosome 18 with SSX1, SSX2 or SSX4 gene on chromosome X. SS predominantly arises in the soft tissue around large joints of the lower extremities. Other sites include head and neck, thorax, heart or pericardium, abdomen and pelvis. Exceedingly rare cases have been reported in an intramuscular location at the shoulder. The cytological diagnosis of a monophasic synovial sarcoma can be challenging, due to the wide spectrum of differential diagnosis. Here we describe a rare case of an intramuscular monophasic synovial sarcoma diagnosed by an integrated cytological and molecular approach.
关键词： Synovial sarcoma,Intramuscular,Fine needle cytology,Monophasic synovial sarcoma,Fluorescence in situ hybridization
更新于2025-09-10 09:29:36
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[Methods in Molecular Biology] Prenatal Diagnosis Volume 1885 || Rapid Prenatal Aneuploidy Screening by Fluorescence In Situ Hybridization (FISH)
摘要： The most common aneuploidies observed in prenatal diagnostics in the second trimester are trisomies of the chromosomes 13, 18 or 21 and gonosomal abnormalities. Rapid detection of these aneuploidies after amniocentesis is possible by ?uorescence in situ hybridization (FISH) utilizing centromeric or locus-speci?c probes. FISH aneuploidy screening results in uncultured amniocytes are available within 24 h or less. Operators should be aware that there are possible pitfalls in connection with the commercially available probe sets and in result interpretation in general and thus proceed with appropriate caution. Here, we explain how rapid prenatal aneuploidy screening is performed using the Food and Drug Administration (FDA-) approved Aneu Vysion kit (ABBOTT/Vysis) and a review is given of drawbacks and opportunities of the method.
关键词： Molecular cytogenetics,Prenatal diagnosis,Interphase FISH,Fluorescence in situ hybridization (FISH),Pitfalls
更新于2025-09-10 09:29:36
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Molecular Diagnostics in Cytopathology (A Practical Handbook for the Practicing Pathologist) || Fluorescence In Situ Hybridization (FISH) Testing in Urinary Tract Cytology
摘要： Fluorescence in situ hybridization (FISH) allows for visualization of specific DNA sequences and can, therefore, be used for quantitation of chromosomes and genes, including aneusomies, chromosomal deletions, or amplifications. The commercial assay UroVysion? (Vysis, Inc., Downers Grove, IL, USA) has made the FISH technique available for routine use in hospital cytology laboratories, but most UroVysion? tests are performed at large reference laboratories. The UroVysion? assay is composed of four single-stranded fluorescently labeled nucleic acid probes – three chromosome enumeration probes (CEP) for the chromosomes 3, 7, and 17 and the single locus-specific identifier (LSI) probe 9p21. The DNA probes are directly labeled with the four different fluorescent dyes SpectrumRed (CEP3), SpectrumGreen (CEP7), SpectrumAqua (CEP17), and SpectrumGold (LSI 9p21).
关键词： UroVysion?,locus-specific identifier,Fluorescence in situ hybridization,urothelial carcinoma,urinary cytology,FISH,chromosome enumeration probes
更新于2025-09-10 09:29:36
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Fluorescence in?situ hybridization comparison of the prognostic factors in adult and pediatric acute lymphoblastic leukemia: A retrospective analysis of 282?cases
摘要： Acute lymphoblastic leukemia (ALL) affects both children and adults. However, the prognosis of the two cohorts is quite different. The present aim was to review and evaluate one potential cause of why survival is poorer in adult ALL than pediatric ALL via fluorescence in situ hybridization (FISH). Clinical significant features were analyzed in 282 ALL cases. FISH was performed to study mixed lineage leukemia (MLL) translocation and the Philadelphia (Ph) chromosome in newly diagnosed patients, and was used to detect trisomy 4 or 10 and the translocation ETS leukemia-acute myeloid leukemia 1 (TEL-AML1) fusion gene. The overall survival/event-free survival (OS/EFS) outcome of adult ALL and pediatric ALL was analyzed using Kaplan-Meier analysis. Adult ALL had a higher median leukocyte count and lower hemoglobin level than pediatric ALL. FISH revealed that Ph positivity (Ph+) was associated with the high-risk feature of older age. In pediatric ALL, trisomy 4 or 10 was present in 71/207 cases (34.3%), while the TEL-AML1 fusion gene was present in 16/207 cases (7.7%). By contrast, there were very few such positive cases in adult ALL. Survival analysis revealed that, in adult ALL, the 3-year OS and EFS rates were higher in the Ph-negative group than in the Ph+ group. Adult or pediatric lymphoblastic leukemia, ALL is an independent prognostic factor of OS. The present analysis of the clinical and biological features between adult and pediatric ALL indicates that adult ALL has a poorer prognosis than pediatric ALL based on Ph+ status and presence of trisomy 4 or 10. Ph+ ALL is an independent prognosis factor of ALL. FISH may serve an important role in the comparison of prognostic factors in adult and pediatric ALL.
关键词： fluorescence in situ hybridization,poor survival,pediatric acute lymphoblastic leukemia,adult acute lymphoblastic leukemia,Philadelphia+ acute lymphoblastic leukemia
更新于2025-09-09 09:28:46