in suite hybridization (objective, applications, protocol)

1. In situ Hybridization
Muhammad Noman Naseem
D.V.M, M.Phil. (Pathology)
Cellular And Molecular Pathology
nomannaseem97@yahoo.com
Mob# +92-3226302788

2. INTRODUCTION
• In situ hybridization was invented by Joseph G.
Gall.
• In suite hybridization is biological assay (like
ELISA, PCR) for molecular diagnosis.
• In situ hybridization (ISH) is a powerful
technique for localizing specific nucleic acid
targets within fixed tissues and cells, allowing
you to obtain information about gene
expression and genetic loci.

3. Continue……..
In situ hybridization (ISH) is a type of
hybridization that uses a labeled complementary
DNA or RNA strand (i.e., probe) to localize a
specific DNA or RNA sequence in a portion or
section of tissue (in situ), or, if the tissue is small
enough (e.g. plant seeds, Drosophila embryos),
in the entire tissue (whole mount ISH), in cells
and in circulating tumor cells (CTCs).

4. Continue……..
• 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.

5. Continue……..
• In situ hybridization is used to reveal the
location of specific nucleic acid sequences on
chromosomes or in tissues, a crucial step for
understanding the organization, regulation
and function of genes.

6. Applications of ISH
• Prenatal test during pregnancy
• Conventional prenatal tests for chromosomal
abnormalities such as Down Syndrome rely on
analyzing the number and appearance of the
chromosomes the karyotype.
• Molecular diagnostics tests such as
microarray comparative genomic hybridisation
test a sample of DNA instead, and because of
cell-free DNA in plasma, could be less invasive.

7. Application Continue……
• Treatment;
• Pharmacogenomics
• Some of a patient's s—slight differences in their
DNA—can help predict how quickly they will
metabolize particular drugs; this is called
pharmacogenomics.
• For example, the enzyme CYP2C19 metabolizes
several drugs, such as the anti-clotting agent
Clopidogrel, into their active forms. Some patients
possess polymorphisms in specific places on the
2C19 gene that make poor metabolisers of those
drugs; physicians can test for these polymorphisms
and find out whether the drugs will be fully
effective for that patient

8. Application Continue……
• Pathogenomics
• Molecular diagnostics are used to identify
infectious diseases such as chlamydia,
influenza virus and tuberculosis; or specific
strains such as H1N1 virus.

9. Types of ISH
• Today there are two basic ways to visualize your
RNA and DNA targets in situ—
• fluorescence (FISH) and
• chromogenic (CISH) detection.
• Characteristics inherent in each method of
detection have made FISH and CISH useful for
very distinct applications. While both use a
labeled, target-specific probe that is hybridized
with the sample, the instrumentation used to
visualize the samples is different for each
method.

10. Types Continue
• Chromogenic in situ hybridization (CISH)
enables you to gain genetic information in the
context of tissue morphology.
• Fluorescence in situ hybridization (FISH)
enables you to assay multiple targets
simultaneously and visualize co-localization
within a single specimen.

11. In Situ Hybridization Protocol
General procedure and tips for in situ
hybridization using antibody detection.
 In situ hybridization indicates the localization
of gene expression in their cellular
environment. A labeled RNA or DNA probe can
be used to hybridize to a known target mRNA
or DNA sequence within a sample.
 This labeled RNA or DNA probe can then be
detected by using an antibody to detect the
label on the probe. The probes can therefore
be used to detect expression of a gene of
interest and the location of the mRNA.

12. How To Store a Sample
• Preserving DNA is easy because it is a highly
stable molecule.
• However, preserving RNA is much more difficult
due to presence of RNase enzyme. This may be
found on glassware, in reagents and on the
operator and their clothing.
• RNase will quickly destroy any RNA in the cell or
the RNA probe itself.
• Therefore, users must ensure they use sterile
techniques, gloves, and solutions to prevent
RNase from contaminating and destroying the
probe or tissue RNA.

13. Storage Continue…….
• General sample storage when using frozen
sections:
• For good results on older slides, the slides
should not be stored dry at room
temperature. They should be stored either in
100% ethanol at -20°C, or in a plastic box
covered in saran wrap at -20°C or -80°C. Slides
stored in this way can be used for several
years.

14. Choice of probe
• RNA probes:
RNA probes should be between 250 to 1500
bases in length. Probes approximately 800 bases
long exhibit the highest sensitivity and specificity.
• DNA probes:
DNA probes can also provide high sensitivity for
in situ hybridization.. However, they do not
hybridize as strongly to the target mRNA
molecules as RNA probes.

15. DIG (Digoxigenin) labeled RNA probe
In situ hybridization protocol
• The protocol shown here describes the use of DIG
labeled single stranded RNA probes to detect expression
of the gene of interest in paraffin embedded sections.
• It is a highly sensitive technique.
 Step (1) Deparaffinization
• If using formaldehyde fixed paraffin embedded sections.
• Before proceeding with the staining protocol, the slides
must be deparaffinized and rehydrated.
• Incomplete removal of paraffin can cause poor staining of
the section

16. CONTINUE…..
• Place the slides in a rack, and perform the following washes:
1) Xylene: 2 x 3 min
2 ) Xylene 1:1 with 100% ethanol: 3 min
3 ) 100% ethanol: 2 x 3 min
4 ) 95% ethanol: 3 min
5 ) 70 % ethanol: 3 min
6 ) 50 % ethanol: 3 min
7 ) Running cold tap water to rinse
• Keep the slides in the tap water until ready to perform antigen
retrieval.
• t no time from this point onwards should the slides be
allowed to dry.
• Drying out will cause non-specific antibody binding and
therefore high background staining.

17. ISH Protocol Continue….
 Step(2) Antigen retrieval
• (antigen retrieval A process in which tissues are heated with a microwave oven,
steam (pressure) cooker, autoclave or ultrasound, which causes antigens that are
difficult or impossible to stain in formalin-fixed paraffin sections of tissue to
become readily stainable by standard immunohistochemical methods.)
• Digest with 20 μg/ml proteinase K in pre-warmed 50 mM
Tris for 10 to 20 min at 37°C. The time of incubation and
concentration of proteinase K may require some
optimization.
• The concentration of proteinase K and the incubation time for this step
will require optimization. We recommend trying a proteinase K
titration experiment to determine the optimal conditions. Insufficient
digestion will result in a reduced hybridization signal. Over digestion
will result in poor tissue morphology, making localization of the
hybridization signal very difficult. The concentration of proteinase K
needed will vary depending upon the tissue type, length of fixation, and
size of tissue.

18. ISH Protocol Continue….
 Step(3) Rinse slides 5 times in distilled water
 Step(4) Immerse slides in ice cold 20% (v/v)
acetic acid for 20 sec. This will permeabilize the
cells to allow access to the probe and the
antibody.
 Step(5) Dehydrate the slides by washing for
approximately 1 min each wash in 70% ethanol,
95% ethanol and 100% ethanol then air dry.
 Step(6) Add 100 μl of hybridization solution to
each slide.

19. ISH Protocol Continue….
 Step(7) Incubate the slides for 1 hr in a humidified
hybridization chamber at the desired hybridization
temperature. Typical hybridization temperatures
range between 55 and 62°C .(The optimal hybridization
temperature for the probe depends on the percentage of
bases present in the target sequence. The concentration of
cytosine and guanine in the sequence are an important factor)
 Step(8) Dilute the probes in hybridization solution in
PCR tubes. Heat at 95°C for 2 min in a PCR block. This
will denature the RNA or DNA probe. Chill on ice
immediately to prevent reannealling.

20. ISH Protocol Continue….
 Step(8) Drain off the hybridization solution.
Add 50 to 100 μl of diluted probe per section
(ensure the entire sample is covered).
Incubate in the humidified hybridization
chamber at 65°C overnight. While incubating,
the sample on the slide can be covered with a
cover slip to prevent evaporation.

21. ISH Protocol Continue….
 Step(9) Stringency washes:
Solution parameters such as temperature, salt
and/or detergent concentration can be manipulated
to remove any non-identical interactions (i.e. only
exact sequence matches will remain bound).
• This step removes non-specific and/or repetitive DNA
/ RNA hybridization. The less concentrated the salt
solution and the longer the duration of the wash and
the temperature, the higher the stringency and the
more DNA / RNA will be removed

22. ISH Protocol Continue….
• . Step(10)Wash twice in MABT (maleic acid
buffer containing Tween 20) for 30 min at room
temperature.
• MABT is gentler than PBS and is more suitable for
nucleic acid detection
• Step(11) Dry the slides.
• Step(12) Transfer to a humidified chamber and
add 200 μl blocking buffer to each section
(MABT + 2% BSA, milk or serum). Block for 1 to 2
hours, at room temperature.

23. ISH Protocol Continue….
 Step(13) Drain off the blocking buffer. Add
the anti-‘label’ antibody at the required
dilution in blocking buffer. Check the antibody
datasheet for a recommended
concentration/dilution. Incubate for one to
two hours at room temperature.

24. ISH Protocol Continue….
 Step(14) Wash slides 5 times with MABT, 10 min for
each wash, at room temperature.
 Step(15) Wash the slides 2 times for 10 min each at
room temperature with pre-staining buffer.
(100 mM Tris pH 9.5, 100 mM NaCl, 10 mM MgCl2).
 Step(16) Fluorescence – please proceed to step 18
Other – return slides to humidified chamber and follow
manufacturer’s instructions for color development.

25. ISH Protocol Continue….
 Step(17) Fluorescence ……….
Other – return slides to humidified chamber and
follow manufacturer’s instructions for color
development.
 Step(18) Rinse slides in distilled water.
 Step(19) Air dry the slides for around 30 min.
Wash in 100% ethanol, then air dry thoroughly.
 Step(20) Mount using DePeX mounting solution.