In situ hybridization is a powerful technique for identifying specific mRNA species within individual cells in tissue sections, providing insights into physiological processes and disease pathogenesis. However, in situ hybridization requires that many steps be taken with precise optimization for each tissue examined and for each probe used. https://www.creative-bioarray.com/Services/In-Situ-Hybridization-ISH.htm

1. In Situ
Hybridization

2. CONTENT
NO.1 ISH
NO.2 CISH
NO.3 FISH
NO.4 General Procedure

3. In Situ Hybridization
In Situ Hybridization (ISH) is a
technique that allows for
precise localization of a specific
segment of nucleic acid within
a histologic section. The
underlying basis of ISH is that
nucleic acids, if preserved
adequately within a histologic
specimen, can be detected
through the application of a
complementary strand of
nucleic acid to which a reporter
molecule is attached.
Introduction
In Situ Hybridization

4. Probes
Double-stranded DNA
(dsDNA) probes
Single-stranded DNA
(ssDNA) probes
RNA probes (riboprobes)
Synthetic oligonucleotides
(PNA, LNA)
Probes - ISH

5. Probe types and their pros and cons
Probe Types Advantages Disadvantages
Double-stranded DNA (dsDNA)
probes
Stable, available, easier to obtain Self-hybridize, less sensitive, need
denaturation before hybridization
Single-stranded DNA (ssDNA)
probes
Stable, easier to work with, more
specific, resistant to RNases,
better tissue penetration, without
self-hybridize
Time consuming, expensive
RNA probes (riboprobes) Higher thermal stability, better
tissue penetration, more specific,
low background noise by RNase
Sensitive to RNases
Synthetic oligonucleotides Economical, stable, available,
easier to work with, more specific,
resistant to RNases, better tissue
penetration, better reproducibility
Know the information of
nucleotide sequence
Probes - ISH

6. Labeling Techniques - ISH
Probe Labeling and Signal Detection
✓ 32P
✓ 35S
✓ 3H
✓ Biotin
✓ Digoxigenin
✓ Fluorescent dye
(FISH)
Radioactive isotopes Non-radioactive labels

8. Chromogenic In Situ Hybridization (CISH)
CISH enables examination of gene amplification,
gene deletion, chromosomal translocations, and
chromosomal number. This approach uses
conventional peroxidase or alkaline phosphatase
reactions using bright-field microscopy on
tissues fixed by formalin and embedded in
paraffin. These peroxidase or alkaline
phosphatase-labeled reporter antibodies
interact with a hybridized DNA probe, and are
then observed with an enzymatic reaction. Tissue
morphology and genetic abnormalities can be
viewed at the same time with CISH.
Introduction of CISH

9. Variations - CISH
SISH uses a similar method as
CISH, but a silver precipitate is
the end product rather than a
brown product.
Silver-enhanced in situ
hybridization (SISH)

10. 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0
DuoCISH is a variation of CISH
that addresses the need for
two different probes on the
same slide. It is a well-
established technique for HER-
2/neu amplification detection
even though it is sometimes
reported to be less effective
than FISH.
DuoCISH
Variations - CISH

11. Medical Applications
Gene amplification
CISH is frequently applied to assess gene
amplification, such as HER-2/neu status in
breast cancer samples.
Chromosomal rearrangements
CISH is also used for detection of chromosomal
rearrangements and fusions, such as the fusion
of ALK tyrosine kinase domain with the
promoter and 5’ region of EML4 in lung cancer.
Papillomavirus infections
Apart from cancers, CISH has also been shown
to be useful in detecting human papillomavirus
infections.
Chromogenic In Situ Hybridization (CISH)

12. Fluorescence In Situ Hybridization (FISH)
Fluorescence in
situ hybridization (FISH) is
a molecular
cytogenetic technique that
uses fluorescent
probes that bind to only
those parts of a nucleic
acid sequence with a high
degree of
sequence complementarity.

13. Single-molecule RNA FISH is a method of detecting
and quantifying mRNA and other long RNA
molecules in a thin layer of tissue sample.
Single-molecule RNA FISH assays can be performed
in simplex or multiplex, and can be used as a follow-
up experiment to quantitative PCR, or imaged
simultaneously with a fluorescent antibody assay.
The technology has potential applications in cancer
diagnosis, neuroscience, gene expression analysis,
and companion diagnostics.
Single-molecule RNA FISH
Variations - FISH

14. Variations - FISH
In an alternative technique
to interphase or metaphase
preparations, fiber FISH, interphase
chromosomes are attached to a slide in
such a way that they are stretched out
in a straight line, rather than being
tightly coiled, as in conventional FISH,
or adopting a chromosome
territory conformation, as in interphase
FISH. This is accomplished by applying
mechanical shear along the length of
the slide, either to cells that have been
fixed to the slide and then lysed, or to a
solution of purified DNA.
Fiber FISH

15. Q-FISH
MA-FISH
Variations - FISH
Microfluidics-assisted FISH (MA-FISH) uses a
microfluidic flow to increase DNA hybridization
efficiency, decreasing expensive FISH probe
consumption and reduce the hybridization time.
MA-FISH is applied for detecting
the HER2 gene in breast cancer tissues.
Q-FISH combines FISH with PNAs and
computer software to quantify
fluorescence intensity. This technique is
used routinely in telomere length research.

16. Hybrid Fusion FISH (HF-FISH) uses
primary additive excitation/emission
combination of fluorophores to
generate additional spectra through a
labeling process known as dynamic
optical transmission (DOT). Three
primary fluorophores are able to
generate a total of 7 readily detectable
emission spectra as a result of
combinatorial labeling using DOT.
Hybrid Fusion FISH enables highly
multiplexed FISH applications that are
targeted within clinical oncology panels.
Hybrid Fusion-FISH
Variations - FISH

17. Species Identification
Comparative Genomic Hybridization
Virtual Karyotype
Spectral Karyotype
Medical Applications -FISH

18. General Procedure
- Select appropriate BAC for target of interest, extract, and amplify DNA
- Label probe with biotin or digoxigenin using random priming or nick translation
Probe
Design
- Fix tissue to a glass slide using paraffin
- Wash and heat slide several times to remove paraffin from surface
- Pepsin digestion to ensure access to target DNA sequence
Tissue
Prep
- Add 10-20 uL probe to tissue, cover slide, and seal coverslip
- Heat slide to 97℃ for 5-10 min to denature DNA
- Place slide in 37℃ oven overnight for probe to hybridize
Hybridization
- Add a blocker to bind nonspecific binding sites
- Add hydrogen peroxide to suppress endogenous peroxidase activity
Blocking
- - If digoxigenin is the label: add anti-digoxigenin fluorescein primary antibody
followed by a HRP-conjugated anti-fluorescein secondary antibody
- - If biotin is the label: HRP-conjugated streptavidin is used for detection
- - Add DAB which is converted to a brown precipitate by HRP
Probe
Detection

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