In situ hybridization (ISH) and fluorescence in situ hybridization (FISH) are methods used to detect nucleic acid sequences in intact cells or tissue sections via specific probes. The choice of probe type and labeling technique is crucial for effective results, with various types available including DNA and RNA probes. These techniques are widely applied in fields like pathology, developmental biology, and physical mapping.

1. In situ Hybridization (ISH)
&
Fluorescence in situ Hybridization (FISH)
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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.
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3. Principle
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ISH works by using a tagging cDNA or probe that will hybridize with the
target DNA sequence

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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.

5. Probe types
Four basic probe types
Stable, available, easier to obtain
dsDNA probes
Stable, easier to work with, more
specific, resistant to RNases, better
tissue penetration, without self-
hybridize
ssDNA probes
Economical, stable, available, easier to work with, more specific, resistant to
RNases, better tissue penetration, better reproducibility.
Synthetic oligonucleotides probes
Higher thermal stability, better tissue
penetration, more specific, low
background noise by RNase
RNA probes
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6. Labeling Probe
Labeling techniques
Radioactive isotopes
• 32
P
• 35
S
• 3
H
Non-radioactive labels
• biotin
• digoxigenin
• fluorescent dye (FISH)
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7. Protocol outline
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Sample preparation
Probe preparation
Denaturation & Hybridization
Detection

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Procedure
Sample Preparation
Embedded
Sectioned

9. Procedure
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Denaturation & hybridization
Permeablization
Probe added

10. Procedure
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Denaturation & hybridization
Washes and detection

11. Procedure
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Detection
NonradioactiveprobeRadioactiveprobe

12. Application
 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
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In situ hybridization has been widely used for research applications

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