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Anatomical Record (Hoboken, N.J. : 2007) Aug 2014In situ hybridization is a technique that is used to detect nucleotide sequences in cells, tissue sections, and even whole tissue. This method is based on the... (Review)
Review
In situ hybridization is a technique that is used to detect nucleotide sequences in cells, tissue sections, and even whole tissue. This method is based on the complementary binding of a nucleotide probe to a specific target sequence of DNA or RNA. These probes can be labeled with either radio-, fluorescent-, or antigen-labeled bases. Depending on the probe used, autoradiography, fluorescence microscopy, or immunohistochemistry, respectively, are used for visualization. In situ hybridization is extensively used in research, as well as clinical applications, especially for diagnostic purposes. This review discusses the basic technique of in situ hybridization. The standard in situ hybridization process is reviewed, and different types of in situ hybridization, their applications, and advantages and disadvantages are discussed.
Topics: Autoradiography; Humans; In Situ Hybridization; Isotope Labeling; Microscopy, Fluorescence; Paraffin Embedding; RNA Probes; Tissue Fixation
PubMed: 24810158
DOI: 10.1002/ar.22944 -
Development (Cambridge, England) Jun 2018hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse...
hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging - analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry - analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging - digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples.
Topics: Animals; Chick Embryo; Escherichia coli; Flow Cytometry; Gene Expression Profiling; Humans; Imaging, Three-Dimensional; In Situ Hybridization; RNA Probes; RNA, Messenger; Reproducibility of Results; Subcellular Fractions
PubMed: 29945988
DOI: 10.1242/dev.165753 -
Molecules (Basel, Switzerland) Apr 2020Fluorescence in situ hybridization (FISH) is a standard technique used in routine diagnostics of genetic aberrations. Thanks to simple FISH procedure is possible to... (Review)
Review
Fluorescence in situ hybridization (FISH) is a standard technique used in routine diagnostics of genetic aberrations. Thanks to simple FISH procedure is possible to recognize tumor-specific abnormality. Its applications are limited to designed probe type. Gene rearrangements e.g., , reflecting numerous translocational partners, deletions of critical regions e.g., 1p and 19q, gene fusions e.g., , genomic imbalances e.g., 6p, 6q, 11q and amplifications e.g., are targets in personalized oncology. Confirmation of genetic marker is frequently a direct indication to start specific, targeted treatment. In other cases, detected aberration helps pathologists to better distinguish soft tissue sarcomas, or to state a final diagnosis. Our main goal is to show that applying FISH to formalin-fixed paraffin-embedded tissue sample (FFPE) enables assessing genomic status in the population of cells deriving from a primary tumor or metastasis. Although many more sophisticated techniques are available, like Real-Time PCR or new generation sequencing, FISH remains a commonly used method in many genetic laboratories.
Topics: Biomarkers, Tumor; Chromosome Painting; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Molecular Probes; Neoplasms; Precision Medicine; Reproducibility of Results
PubMed: 32316657
DOI: 10.3390/molecules25081864 -
Nature Neuroscience Dec 2021The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors, and its dysfunction has been linked to many neural disorders. However, the...
The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors, and its dysfunction has been linked to many neural disorders. However, the molecular, cellular and anatomic heterogeneity underlying its functional diversity remains incompletely understood. In this study, we generated a cell census of the mouse NAc using single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization, revealing a high level of cell heterogeneity in this brain region. Here we show that the transcriptional and spatial diversity of neuron subtypes underlie the NAc's anatomic and functional heterogeneity. These findings explain how the seemingly simple neuronal composition of the NAc achieves its highly heterogenous structure and diverse functions. Collectively, our study generates a spatially resolved cell taxonomy for understanding the structure and function of the NAc, which demonstrates the importance of combining molecular and spatial information in revealing the fundamental features of the nervous system.
Topics: Animals; Brain; In Situ Hybridization, Fluorescence; Mice; Neurons; Nucleus Accumbens
PubMed: 34663959
DOI: 10.1038/s41593-021-00938-x -
Nucleic Acids Research Nov 2020Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional spatial dimension to...
Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional spatial dimension to investigate multiplexed gene expression, cell types, disease architecture or even data driven discoveries. In situ sequencing (ISS) method based on padlock probes and rolling circle amplification has been used to spatially resolve gene transcripts in tissue sections of various origins. Here, we describe the next iteration of ISS, HybISS, hybridization-based in situ sequencing. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes for high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. HybISS design allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a targeted amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.
Topics: Animals; Brain; Computational Biology; Humans; In Situ Hybridization, Fluorescence; Mice; RNA; Single-Cell Analysis; Transcriptome
PubMed: 32990747
DOI: 10.1093/nar/gkaa792 -
Methods (San Diego, Calif.) Jun 2014The eye has been one of the most intensively studied organs in Drosophila. The wealth of knowledge about its development, as well as the reagents that have been... (Review)
Review
The eye has been one of the most intensively studied organs in Drosophila. The wealth of knowledge about its development, as well as the reagents that have been developed, and the fact that the eye is dispensable for survival, also make the eye suitable for genetic interaction studies and genetic screens. This article provides a brief overview of the methods developed to image and probe eye development at multiple developmental stages, including live imaging, immunostaining of fixed tissues, in situ hybridizations, and scanning electron microscopy and color photography of adult eyes. Also summarized are genetic approaches that can be performed in the eye, including mosaic analysis and conditional mutation, gene misexpression and knockdown, and forward genetic and modifier screens.
Topics: Animals; Developmental Biology; Drosophila; Eye; Gene Expression Regulation, Developmental; Humans; In Situ Hybridization; Microscopy, Electron, Scanning; Mutation
PubMed: 24784530
DOI: 10.1016/j.ymeth.2014.04.007 -
PloS One 2015In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across...
In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.
Topics: Animals; Brain; Immunohistochemistry; In Situ Hybridization; Male; Neurons; Primary Cell Culture; Rats
PubMed: 25794171
DOI: 10.1371/journal.pone.0120120 -
Cancer Metastasis Reviews Mar 2015Human epidermal growth factor receptor 2 (HER2) amplification/overexpression is an effective therapeutic target in breast and gastric cancer. Although HER2 positivity...
Human epidermal growth factor receptor 2 (HER2) amplification/overexpression is an effective therapeutic target in breast and gastric cancer. Although HER2 positivity has been reported in other malignancies, previous studies generally focused on one cancer type, making it challenging to compare HER2 positivity across studies/malignancies. Herein, we examined 37,992 patient samples for HER2 expression (+/- amplification) in a single laboratory. All 37,992 patients were tested by immunohistochemistry (IHC); 21,642 of them were also examined for HER2 amplification with either fluorescent in situ hybridization (FISH) (11,670 patients) or chromogenic in situ hybridization (CISH) (9,972 patients); 18,262 patients had tumors other than breast or gastric cancer. All tissues were analyzed in a Clinical Laboratory Improvement Amendments (CLIA) laboratory (Caris Life Sciences) at the request of referring physicians. HER2 protein overexpression was found in 2.7 % of samples. Over-expressed HER2 was detected predominantly in malignancies of epithelial origin; for cancers derived from mesenchyme, neuroendocrine tissue, central nervous system, and kidney, HER2 expression and amplification were remarkably rare or non-existent. Bladder carcinomas, gallbladder, extrahepatic cholangiocarcinomas, cervical, uterine, and testicular cancers showed HER2 positivity rates of 12.4, 9.8, 6.3, 3.9, 3.0, and 2.4 %, respectively. HER2 overexpression and/or amplification is frequently found across tumor types. These observations may have significant therapeutic implications in cancers not traditionally thought to benefit from anti-HER2 therapies.
Topics: Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization; In Situ Hybridization, Fluorescence; Neoplasms; Receptor, ErbB-2
PubMed: 25712293
DOI: 10.1007/s10555-015-9552-6 -
Archives of Pathology & Laboratory... Nov 2018To update key recommendations of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human epidermal growth factor receptor 2 (HER2)...
Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update.
PURPOSE.—
To update key recommendations of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline.
METHODS.—
Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations.
RECOMMENDATIONS.—
Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in >10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not "must") be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended workup for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 ( HER2/chromosome enumeration probe 17 [CEP17] ratio ≥2.0; average HER2 copy number <4.0 signals per cell), ISH group 3 ( HER2/CEP17 ratio <2.0; average HER2 copy number ≥6.0 signals per cell), and ISH group 4 ( HER2/CEP17 ratio <2.0; average HER2 copy number ≥4.0 and <6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results.
Topics: Female; Humans; Biomarkers, Tumor; Breast Neoplasms; Immunohistochemistry; In Situ Hybridization; Medical Oncology; Receptor, ErbB-2; United States; Systematic Reviews as Topic
PubMed: 29846104
DOI: 10.5858/arpa.2018-0902-SA -
The Journal of Neuroscience : the... Jan 2020Without question, molecular biology drives modern neuroscience. The past 50 years has been nothing short of revolutionary as key findings have moved the field from... (Review)
Review
Without question, molecular biology drives modern neuroscience. The past 50 years has been nothing short of revolutionary as key findings have moved the field from correlation toward causation. Most obvious are the discoveries and strategies that have been used to build tools for visualizing circuits, measuring activity, and regulating behavior. Less flashy, but arguably as important are the myriad investigations uncovering the actions of single molecules, macromolecular structures, and integrated machines that serve as the basis for constructing cellular and signaling pathways identified in wide-scale gene or RNA studies and for feeding data into informational networks used in systems biology. This review follows the pathways that were opened in neuroscience by major discoveries and set the stage for the next 50 years.
Topics: Animals; CRISPR-Cas Systems; Exocytosis; Gene Expression Regulation; Gene Transfer Techniques; Genes, Reporter; History, 20th Century; History, 21st Century; Humans; In Situ Hybridization; Microscopy; Molecular Biology; Nerve Tissue Proteins; Neurosciences; PDZ Domains; Polymerase Chain Reaction; Protein Engineering; RNA; Recombinant Proteins; Sequence Analysis, DNA
PubMed: 31630114
DOI: 10.1523/JNEUROSCI.0743-19.2019