Advanced Research Techniques

In Basic Medical Sciences

“ In Situ ”
Hybridization
Techniques
Associate Professor Dr.
Özhan Eyigör
Uludag University College of Medicine
Department of Histology & Embryology
 Hybridization

• Hybridization:
In solution – in vitro
On cell preparations or tissue sections – in situ
On nitrocellulose membranes
• Southern Blotting: DNA
• Northern Blotting: RNA
 What is In Situ Hybridization

• In situ hybridization is a powerful technique that

enables the detection of specific mRNA species
within individual cells in tissue sections and can
provide invaluable insights into physiological
processes and disease pathogenesis.

– Preservation of target mRNA

 ‘In Situ’ Hibridizasyon
(hibridizasyon histokimyası, sitolojik hibridizasyon)

• Morphologic demonstration of specific RNA (or DNA)

sequences on:
• Tissues
• Cell cultures
• Chromosome prepapations

• In theory: The principle of the technique depends on the

hybridization of labeled single-strand DNA (or RNA)
sequences (= probe =complimentary secuence) to the
cellular RNA or DNA.
• 1969: Buongiorno-Nardelli and Amaldi –
Nature
• 1969: Gall and Pardue – PNAS
• 1969: John, Birnstiel and Jones - Nature
 Usage of In situ Hybridization

• Localization of the DNA sequences

• Bacterial or viral DNA or RNA detection
• Producing chromozomal genetic maps
• Analysis of chromozomal errors
• Detection of the differences of genetic expressions
• Detection of mRNA expression and localization
• Detection of virus or bacteria in tissues

• Developmental biology
• Cell biology
• Histology
• Genetics
• Microbiology
• Pathology
 Types of In situ Hybridization

• Radioactive in situ hybridization :

• Sulphur 35 (S35)
• Phosphor 32, 33 (P32, P33)

• Non-Radioactive in situ hybridization

• Digoxigenin
• Biotin
• Dinitrophenil
• Fluorochroms
 The Aim

• To cause least possible damage on the tissue,

• Not to block the probe to pass into the cell,
• To preserve the RNA or DNA,
• To avoid non-specific binding as possible,
• To prevent contamination (especially RNase)
 Tissue Preparation

• The tissue must be taken out of the animal as fast as

possible to avoid the break down of the RNA or DNA.

• Must be fixed or freezed as fast as possible in order to

avoid the effects of RNase on the RNA.

• Sectioning: Cryostat (frozen) sections are commonly used.

Usually the thickness is about 10-15 microns. Paraffin or
resin sections can also be used but not recommended.
 Hybridization Protocol

Three Major Steps:

• Pre-hybridization step: The tissue is

prepared for the hybridization.

• Hybridization.

• Post-hybridization step: The hybridization

signal on the tissue is made visible.
 Prehybridization
A) Preparing the tissue for hybridization

For DNA hybridization: RNA is removed from

the tissue by using RNase.

For RNA hybridization: The solutions,

chemicals ant the tools must be made RNase-free
to avoid RNase contamination.

B) Asetilation
Commonly used for RNA/RNA hybridization.
The aim of the asetilation is to neutralize the
positive charges on the tissue. This
prevents the non-specific binding of the
probe.
 Prehybridization

C) Permeabilization of the tissue:

The aim is to ease the penetration of the probe into the
cells.
1. Protease application: Use of the enzymes which
digest the proteins enhances the permeability.
2. HCl application: Acidic denaturation of the
membranes
3. Detergent application: To decrease the surface
tension and break down the membranes.

D) Neutralization of the endogeneous enzymes.

E) Prehybridization fixation
 Denaturization

• In order to break the double stranded DNA into

single strands for DNA/DNA hybridization.

• Also used for RNA hybridization:

Sometimes the single-stranded RNA probes
curl on itself and appropriate nucleotids bind to
each other to make a partial double strand. This
can be avoided by denaturization.
 Hybridization
• Hybridization with the probe.
• For DNA/DNA hybrids at 370 C
• For RNA/RNA hybrids at 50-550 C

• The concentration of the probe must be determined.

• The contents of the hybridization solution:

1. Probe
2. Formamide (For the specifity of the hybridization)
3. Dextran sulphate (Increases the hybridization ratio)
4. Blocking DNA or tRNA (somon sperm DNA)
5. Sodium dodesyl sulphate (Increases the penetration
of the probe)
6. Bovine serum albumine (Blocks the non-specific
reactions)
7. Salts (to regulate the ionic environment)
 Post-Hybridization washes

• The stringency of the in situ hybridization:

Determines the ratio of the faultless hybridization of
the probe with the target sequence.
The temperature, ionic concentration and the amount
of the formamide are important.

Washing removes the faulty paired hybrids from the tissue.

RNase wash: In the RNA/RNA hybridization unhybridized

probes tend to stick on the tissue. This causes non-specific
results. The RNase breaks down the unhybridized probe.

RNase only effects the single strand RNA, so it does not

break the hybridization complex.
Preparation and Labeling of the Probe
1. Two strand DNA probes
2. Synthetic oligonucleotide probes.
3. Single strand complementary DNA (cDNA) probes.
4. Complementary RNA (cRNA) probes.
*RNA polymerase
One polymerase works on one direction:
Antisense Probe
Other polymerase works on the other direction:
Sense Probe
Antisense probe is used for the hybridization.
Sense probe has the same sequence with the target
nucleotide strand.
*Labeled nucleotid (UTP)
*Labeled probe
 Non-Radioactive labels:
Digoxigenin,
Fluorochromes, Biotin

Radioactive
labels:
P32, P33, S35
 Detection of Hybridization

A) Detection of Non-radioactive Probes:

Immunohistochemical Method: A primary antibody against

the label is used: For example; anti-digoxigenin. The next
steps are the same with immunohistochemical method.

Biotin Avidin Method: If the probe is labeled with biotin,

then avidin is used to visualise the complex.

Systems with direct signaling: Fluorochromes, Enzymes,

and metals (colloidal gold).
 Detection of Hybridization

B) Detection of Non-radioactive Probes :

Autoradiography:

1. Film Autoradiography :
Hybridization slides are put on a specific rontgen film.
This film must be sensitive to the radioactive label
used for hybridization.
The film should be exposed for a certain time.
After developing the hybridization signal is seen as
dark areas on the film.
Film Autoradiography
 Detection of Hybridization

B) Detection of Non-radioactive Probes :

2. Autoradiography on slides:
After hybridization the slides are covered with a
photographic emulsion.
Exposed for a certain time.
After development the hybridization signal is seen as
black dots on the section.
When analyzed with a dark field condenser the signal
is seen as white shiny dots over a dark background.
GluR7 Exression in the Median Eminence

GluR7 GluR7

GluR7 GluR6
Mapping of the Distribution of Glutamate
Receptor mRNA’s in the Hypothalamus
GnRH mRNA Expression in
Hypothalamus
Dual in situ Hybridization – GnRH and GluR5
FISH: Fluorecence in
situ Hybridization
Antisense Method – (Antisense treatment)
 •Southern and Northern Blotting
Southern blotting determines the presence of a particular gene
or DNA sequence within thousands of base pairs in a DNA
molecule. It is widely used for the molecular biology and
recombinant DNA technology studies.

Especially used to determine the presence of a particular DNA

in the studies of:
Gene structure
Gene expression
Genomic organization
Gene transfer
Southern blotting was first introduced by Southern in 1975.

Northern Blotting is used to determine RNA with slight

modification of southern blotting technique.
 •Southern and Northern Blotting
Sometimes it's a bit hard to understand, but there is
humor in science. In the 1970s, E.M. Southern
developed a method for locating a particular
sequence of DNA within a complex mixture. This
technique came to be known as Southern
blotting. In a tongue-in-cheek fashion, those who
used a similar method for locating a sequence of
RNA named it Northern blotting. It is also known
as Northern hybridization or RNA hybridization.
 Principle

The DNA or RNA is hybridized by a complementary

probe.

The probe is labeled with radioactive or non-

radioactive labels.

Following the hybridization the signal is made

visible:
Radioactive label – autoradiography
Biotin label – immunohistochemistry
Flourescent label – flourescence microscopy
 Radioactive Labeling

In the recent years most of the studies used

radioactive labels.

The advantages of radioactive labeling and

autoradiography:
Results are taken so fast
Reliability

Disadvantages:
Not easy to use radioactivity
It is dangerous
 Non-Radioactive Labeling
1. Digoxigenin-anti-digoxigenin system,

2. Horseradish peroksidaz system

3. Biotin-streptavidin system

In order to visualise the complexes of these

systems:
The use of chromogens (colorimetric): Makes
a colored precipitate.
Use of chemiluminergic substances: Emit
light to be observed under direct ultraviolet
light or flourescence microscope.
 Technique

The technique:

• Similar to Western blotting.

• DNA or RNA is seperated on a gel according
to their size by electrophoresis
• Transterred to a nitrocellulose membrane
• Hybridization is employed on the
nitrocellulose membrane
• The hybridization signal is visualised.
 FISH
• FISH (Fluorescent in situ hybridization) is a
cytogenetic technique which can be used to detect
and localize the presence or absence of specific DNA
sequences on chromosomes.
• It uses fluorescent probes which bind only to those
parts of the chromosome with which they show a high
degree of sequence similarity. F
• luorescence microscopy can be used to find out
where the fluorescent probe bound to the
chromosome.
• FISH is often used for finding specific features in
DNA. These features can be used in genetic
counseling, medicine, and species identification.