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ISH and FISH

In situ Hybridization (ISH) is a method that allows to localize and detect nucleic acid sequences within structurally intact cells or morphologically preserved tissues sections. Fluorescence in situ hybridization (FISH) is a kind of ISH which uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity.<br>

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ISH and FISH

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  1. In situ Hybridization (ISH) & Fluorescence in situ Hybridization (FISH) Creative Diagnostics® www.creative-diagnostics.com

  2. Introduction In situ Hybridization (ISH) is a method that allows to localize and detect nucleic acid sequences within structurally intact cells or morphologically preserved tissues sections. Fluorescence in situ hybridization (FISH) is a kind of ISH which uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity. ® Creative Diagnostics ® Creative Diagnostics

  3. Principle ISH works by using a tagging cDNA or probe that will hybridize with the target DNA sequence ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  4. Probe preparation • Probe is critical to in situ hybridization, and a right probe can help you achieve your goals. • Not only the probe types but also the label of probe should you take into account when you choose a probe for in situ hybridization. ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  5. Probe types Four basic probe types ssDNA probes dsDNA probes Stable, easier to work with, more specific, resistant to RNases, better tissue penetration, without self-hybridize Stable, available, easier to obtain RNA probes Synthetic oligonucleotides probes Economical, stable, available, easier to work with, more specific, resistant to RNases, better tissue penetration, better reproducibility. Higher thermal stability, better tissue penetration, more specific, low background noise by RNase ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  6. LabelingProbe Labelingtechniques Radioactive isotopes • 32P • 35S • 3H Non-radioactive labels • biotin • digoxigenin • fluorescent dye (FISH) ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  7. Protocol outline Sample preparation Probe preparation Denaturation & Hybridization Detection ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  8. Procedure Embedded Sectioned Sample Preparation formalin ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  9. Procedure Denaturation & hybridization Probe added Permeablization ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  10. Procedure Denaturation & hybridization Washes and detection ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  11. Procedure Detection Radioactive probe Nonradioactive probe ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  12. Application In situ hybridization has been widely used for research applications • Morphology: Morphology and population structure of microorganisms • Pathology: Pathogen profiling, abnormal gene expression • Developmental biology: Gene expression profiling in embryonic tissues • Karyotyping and phylogenetic analysis: Unique FISH patterns on individual chromosomes, chromosomal aberrations • Physical mapping: Mapping clones on chromosomes and direct assignment of mapped clones to chromosomal regions associated with heterochromatin or euchromatin ® Creative Diagnostics ® ® Creative Diagnostics Creative Diagnostics

  13. Thank You！ For more info please contact us: Phone: 1-631-624-4882 Creative Diagnostics® Email: info@creative-diagnostics.com Go to our website! www.creative-diagnostics.com

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