1. In situ hybridisation (ISH) Fluorescent In situ hybridisation (FISH)
&
Fluorescent In situ hybridisation (FISH)

2. INTRODUCTION:
The word “In situ” comes from Latin meaning “in position”.
By definition it means in the natural or original position.
Hybridization means production of hybrid by cross breading.
In molecular biology hybridization means paring of complementary RNA or DNA to produce a double stranded nucleic acid.
Hybridization method uses a radio or fluorescent labeled DNA or RNA probe that binds to target DNA or RNA of interest, permitting visualization.

3. In situ hybridisation (ISH)
Detection of specific DNA or RNA sequences in tissue sections or
cell preparations using a labeled probe under appropriate
conditions this probe will hybridize the target DNA or
RNA which will be visualized by radioactive or
non radioactive labels incorporated
into the probe.
It is a technique used to examine DNA and RNA in their topographic surroundings.

4. Most important advantages of ISH…
Simplicity of its methodology
Specificity of results obtained
Ease in interpretation of findings
Its applicability on tissue sections (frozen or formalin-fixed, paraffin-embedded) and smears without the need for special specimen collection or processing.

5. HISTORY
First described by almost simultaneously by John et al-& Gall and Pradue.
PRINCIPLE
ISH is the specific annealing of a labelled nucleic acid probe to complementary sequences in fixed tissues followed by visualization of the location of the probe.
ISH –demonstrates specific nucleic acid sequence in their cellular environment.

6. STEPS IN ISH
Probe preparation
Pretreatment of specimens
Hybridization
Detection methods

7. PROBE
PREPARATION

8. PROBES
A probe (a labeled complementary single strand) is incorporated with the DNA/RNA strands of interest.
Strands will anneal with complementary nucleotides bonding back together with their homologous partners when cooled.
Chances of a probe finding a homologous sequence other than the target sequence decreases as the number of nucleotides in the probe increases.

9. TYPES -double strand DNA probe -single strand DNA probe
-single strand RNA probe
-oligonucleotide probes

10. 1. DOUBLE STRANDED DNA PROBE:
Prepared by nick translation, random primer, PCR in the presence of a labelled nucleotide.
Nick translation is a method for incorporating labeled nucleotides into DNA such as an isolated fragment.
The method uses a combination of two enzymes,
Deoxyribonuclease I (DNAse I) which nicks the DNA creating free 3' hydroxyls.
DNA polymerase I, which processively adds nucleotides to the 3' terminal hydroxyl.
This process is called nick translation because the DNA to be processed is treated with DNase to produce single-stranded "nicks." This is followed by replacement in nicked sites by DNA polymerase I, which elongates the 3' hydroxyl terminus, removing nucleotides by 5'-3' exonuclease activity, replacing them with dNTPs.

11. This process is called nick translation because the DNA to be processed is treated with DNase to produce single-stranded "nicks." This is followed by replacement in nicked sites by DNA polymerase I, which elongates the 3' hydroxyl terminus, removing nucleotides by 5'-3' exonuclease activity, replacing them with dNTPs.

12. 1. DOUBLE STRANDED DNA PROBE:
Random priming is a means of labelling DNA fragments whereby,
a mixture of all possible combinations of hexamers, octamers, or nanomers are annealed to denatured DNA.
These small oligonucleotides then act as primers
that allow for synthesis of the complementary DNA strand by the Klenow enzyme and incorporation of both labeled and unlabelled nucleotides.

14. 1. DOUBLE STRANDED DNA PROBE:
Denturated before use.
More effective when the target is abundant – Viral DNA.
Less sensitive than single strand probe,
Because two strands have a tendency to hybridize to each other, thus reducing the concentration of probe available for hybridization to the target.

15. 2. SINGLE STRANDED DNA PROBES:
Single-stranded DNA probes cover a much larger size range (200–500 bp) than oligonucleotide probes.
They can be prepared by…
A primer extension on single-stranded templates by RT-PCR of RNA, or
An amplified primer extension of a PCR-generated fragment in the presence of a single antisense primer, or
Chemical synthesis of oligonucleotides.

16. 2. SINGLE STRANDED DNA PROBES:
PCR-based methods are much easier and probes can be synthesized from small amounts of starting material.
Moreover, PCR allows great flexibility in the choice of probe sequences by the use of appropriate primers.

17. 3. RNA PROBES
RNA probes (cRNA probes or riboprobes) are thermostable and are resistant to digestion by RNases.
These probes are single stranded and are the most widely used in ISH.
RNA probes are generated by in vitro transcription from a linearized template using a promoter for RNA polymerase.
RNA polymerase is used to synthesize RNA complementary to the DNA substrate.

18. Single-stranded probes provide advantages over double-stranded probes such as:
The probe does not self-anneal in solution, so the probe is not exhausted.
Large probe chains are not formed in solution; thus, probe penetration is not affected.
If high sensitivity is required, single-stranded probes should be used

19. 4. OLIGOPROBES Usually shorter 20-40 base pair length.
They are produced synthetically by an automated chemical synthesis.
These probes are resistant to RNases and are small, thus allowing easy penetration into the cells or tissue of interest.
Small size has a disadvantage in that it covers fewer targets.

20. 4. OLIGOPROBES Label should be positioned at the 3′ or the 5′ end.
To increase sensitivity one can use a mixture of oligonucleotides that are complementary to different regions of the target molecule.
Another advantage of oligonucleotide probes is that they are single stranded, therefore excluding the possibility of re-naturation.

21. DNA
RNA
OLIGONUCLEOTIDE
Longer sequences can be produce
Difficult to produce
Insensitive unless used as cocktail of several labeled sequences
Poor Hybridization
High efficiency of hybridization

22. PROPERTIES OF PROBES PROBE CONSTRUCT
Oligonucleotide probes are better than traditional probes because of high specificity, single-stranded and short probe length (10-50 nucleotides)
EFFICIENCY OF LABELLING
Labelling by random priming has been reported to be more efficient than nick translation.
PERCENTAGE OF G-C BASE PAIRS
Higher the content of G-C pairs, the higher the Tm (melting temperature)

23. PROPERTIES OF PROBES RNA VERSUS DNA PROBES
Strength of the probe-target bond decreases in the order of
RNA-RNA, DNA-RNA, DNA-DNA
PROBE LENGTH
Shorter the probe, the better its penetration into cells
SIGNAL DETECTION SYSTEMS
Autoradiography for radioactive labels is reputed to be more sensitive than the immunoenzyme systems.

24. LABELLING OF
PROBE

25. LABELLING OF PROBES
A probe is a labeled fragment of DNA or RNA used to find its complementary sequence or locate a particular clone.

26. There are two methods of probe labelling. They are
LABELLING OF PROBES
There are two methods of probe labelling. They are
DIRECT
Reporter molecules..
1. Enzyme,
2. Radioisotope or
3. Fluorescent marker
are directly attached to the DNA or RNA.
INDIRECT
A hapten..
1. Biotin,
2. Digoxigenin, or
3. Fluorescein
are attached to the probe and detected by a labeled binding protein (typically an antibody).

27. Probes can be labeled with
LABELLING OF PROBES
Probes can be labeled with
RADIOISOTOPES
32 P
35 S
125 I
NON-RADIOACTIVE
Biotin,
Peroxidase
Digoxigenin

28. Isotopic probes are…
Generally more sensitive than non-isotopic ones but are
Less stable,
Require longer processing times
Stringent disposal methods.

29. OLIGONUCLEOTIDE PROBE LABELING
5′-end labelling
The 5′ end of DNA or RNA undergoes direct phosphorylation
of the free 5′-terminal OH groups.
The free 5′-OH substrates can be labeled using T4 polynucleotide kinase.
This method is usually used for radiolabeling.
Non-radiolabels use a covalent linker.

30. OLIGONUCLEOTIDE PROBE LABELING
3′-end labelling
Terminal dexoxynucleotidyl transferase (TdT) is used to add a labeled residue to the 3′ end of a synthetic oligonucleotide that is approximately 14–100 nucleotides in length.
These probes provide excellent specificity but only moderate sensitivity.

31. OLIGONUCLEOTIDE PROBE LABELING
3′ tailing
A tail containing labeled nucleotides is added to the free 3′ end of double- or single-stranded DNA using TdT.
These probes are more sensitive than the 3′-end labeled versions, but can produce more non-specific background.

32. PROBES AND THEIR CHOICE
Double-stranded DNA
Single-stranded DNA
Oligo-deoxyribonucleotides
Probes for
DNA:
Single-stranded complementary RNA, a riboprobe.
Probe for
RNA:

33. PURIFICATION OF LABELED PROBES
There are several methods that can be used to test the purification.
Here is a list of methods that can be used, but it is advisable to follow the manufacturers’ recommendation on their use:
• Sephadex G-50 column
• Sephadex G-50 chromatography
• Selective precipitation

34. Probes without acetylation pre-treatment of the sample
PROBE CONCENTRATION
For DNA probes the concentration of the probe will be 0.5–2 μg/ml.
Oligonucleotide probes can be used with, or without, acetylation.
Probes without acetylation pre-treatment of the sample
will have a concentration of ~50–200 ng/ml and may provide more intense results with minimal background.
For probes with acetylation pre-treatment,
a higher concentration of oligonucleotide probe may be used without incurring non-specific background staining.

35. LENGTH OF PROBE
Optimal probe size for ISH is small fragments of about 200–300 nucleotides.
However, probes may be as small as 20–40 base pairs (bp) or as large as 1000 bp.

36. LENGTH OF PROBE
As probes increase in length, they become more specific.
Longer probes may lead to weaker signal.
They penetrates less efficiently the cross linked tissues.
Extent of weaker signals and penetration depends also on the..
Nature of the tissue,
Choice of fixative and
Whether a pre-treatment has been carried out.

37. PRETREATMENT OF
SPECIMENS

38. PRETREATMENT OF SPECIMENS
Tissue sections must adhere well to specially treated glass slides to avoid loss of tissue during the hybridisation process.
Various "adhesives" are available including..
Poly-L-lysine,
Gelatin chrome alum, and
Aminopropyltriethoxysilane (TESPA).

39. FIXATION
Methanol/acetic acid fixation is recommended for metaphase chromosome spreads.
Cryostat sections may be fixed with 4% formaldehyde (~30 minutes), Bouin’s fixative, or paraformaldehyde vapour fixation.
This fixation also helps to secure the tissue to the slide.
Most commonly, tissue specimens are routinely fixed in 10% buffered formalin, processed overnight in an automatic tissue processor, and embedded in paraffin wax.
Fixation time of 8–12 hours is optimal.

40. SLIDE/SECTION PREPARATION
Sections are cut at 4–6 μm on an alcohol-cleaned microtome using positively charged or hand-coated slides.
Sections are drained well and then air-dried at room temperature.
After deparaffinization, slides are placed in an alcohol-cleaned staining container of DEPC water.
The staining container is then placed in the heated water bath at 23–37°C and held until the start of ISH.
Gloves must be worn to prevent contamination, and all utensils, such as brushes and forceps, should be cleaned with alcohol and kept within the cleaned area designated for ISH.

41. PROTEOLYTIC DIGESTION
The use of formaldehyde-based fixatives prior to paraffin embedding of specimens will mask nucleic acid sequences.
Digestion is a important step when performing ISH.
Digestion improves probe penetration by increasing cell permeability with minimal tissue degradation.

42. HYBRIDAZATION

43. Molecular hybridisation is the process whereby a single-stranded target sequence is annealed to a complementary single-stranded probe to form a double-stranded hybrid.
Prior to hybridisation…
Both the target and the probe, if double-stranded, must be denatured to render them single-stranded and this can be achieved by heat or alkali treatment.

44. Following denaturation…
Single stranded target and probe sequences are incubated in a hybridisation mixture, which provides an optimal environment for re-annealing of single-stranded sequences.

45. HYBRIDIZATION
Hybridization occurs after denaturisation, during cooling, in the presence of a complementary probe, and permits hydrogen bonding of the two strands of nucleic acids.
Probe must form stable hydrogen bonds with the target.
Simultaneously heating the probe and target to high temperatures may increase the consistency and sensitivity of detection.
This can only be met if care is taken to precisely control this step of the ISH procedure.

46. POST-HYBRIDISATION WASHES
Stringency washes after hybridisation aims at decreasing non-specific binding.
However, it is preferable to hybridize stringently rather than wash stringently.

47. DETECTION

48. Direct method or Indirect method
Various methods are available for visualisation of the hybridisation.
Choice of detection system will be principally determined by the probe label used and secondly by the ISH procedure type.
Detection methods can be either
Direct method or
Indirect method

49. There are two methods of probe labelling. They are
LABELLING OF PROBES
There are two methods of probe labelling. They are
DIRECT
Reporter molecules..
1. Enzyme,
2. Radioisotope or
3. Fluorescent marker
are directly attached to the DNA or RNA.
INDIRECT
A hapten..
1. Biotin,
2. Digoxigenin, or
3. Fluorescein
are attached to the probe and detected by a labeled binding protein (typically an antibody).

50. Therefore, indirect methods are preferred.
ENZYMATIC DETECTION
Hybridized probes can be detected by enzymatic reactions that produce a colored precipitate at the site of hybridization.
The most commonly used enzymes for this application are
ALKALINE PHOSPHATASE (AP)
HORSERADISH PEROXIDASE (HRP)
Although these enzymes can be conjugated directly to nucleic acid probes, such enzyme-coupled probes are often inappropriate for ISH to tissue preparations because probe penetration is hampered by the presence of the conjugated enzyme.
Therefore, indirect methods are preferred.

51. DETECTED BY AUTORADIOGRAPHY
Reactions using radioactive labelled probes are detected by autoradiography.
This is based on the emission of fast-electrons or beta particles from the probe.
Beta particles release a large amount of energy when they collide with atoms of an emulsion added to the section on the slide.

52. The excessive energy released reduces ionic silver present in the emulsion to metallic silver.
When this happens, a faithful record of the location of the collision between an electron and the silver ions in the emulsion is produced in the form of a latent image.
This image, when visualised is the indicator of the probe location in the tissue or cell.

54. FLUOROPHORES
Fluorophores can be associated with nucleic acid probes by
Chemical conjugation to the nucleic acid,
Chemical conjugation of the nucleic acid with a nonfluorescent molecule that can bind fluorescent material after hybridization.
The former method is called “direct labelling” and the latter method is called “indirect labelling.”

55. Chemical structures of four common fluorophore classes (A–D)
A. fluoresceins, B. rhodamines, C. cyanines, D. coumarins,

56. INDIRECT METHOD
Indirectly via incorporation of a nucleotide analog carrying a reactive group and subsequent biotinylation/ digoxigenylation.
Resulting Biotin-labeled probes are then detected using streptavidin (KD= M) conjugated with
Horseradish peroxidase (HRP),
Alkaline phosphatase (AP)

57. Due to their moderate size (300 and 400 Da, respectively),
Biotin and Digoxigenin are efficient labels

59. INDIRECT METHOD
Digoxigenylation is typically visualized by HRP- or AP modified
antibodies.

60. INDIRECT METHOD

61. ENZYMATIC REACTION Horseradish peroxidase with either
3-amino-9- ethylcarbazole (AEC) or
3,3′-diaminobenzidine tetra-hydrochloride (DAB) substrates.
AEC forms a red-brown product which is alcohol soluble; therefore aqueous mounting media are required.
Methyl green/blue has been the most often used counterstain.
DAB forms a permanent, insoluble, brown product that is compatible with solvent-based mounting media.

62. ENZYMATIC REACTION Alkaline phosphatase systems can use
5-bromo-4-chloro-3-indolyl phosphate/nitro-blue tetrazolium (BCIP/NBT) or
Fast red.
BCIP/NBT forms a purple/blue alcohol-insoluble stain.
Eosin is a compatible counterstain if a nuclear target is expected, or nuclear fast red if the target is cytoplasmic.
Fast red forms an intense red product which is alcohol soluble and an aqueous mounting media is required.
Methyl green or blue is compatible if a nuclear target is expected, or light hematoxylin if the target is cytoplasmic.

63. MULTIPLE ISH
More than one probe can be applied to the same tissue section to detect different nucleic acid targets.
By using different detection systems with each probe, resulting in different colour end products, visualisation of the different nucleic acid targets can be achieve.

64. In situ hybridisation (ISH)
APPLICATIONS OF
In situ hybridisation (ISH)

65. Infective agents determination of infective agent
This is based on the detection of the infective agent's genome in the tissues or cells studied.
Specific typing of infective agents also have important implications for epidemiological surveys and outbreak investigations.

67. Localisation of active infection
The actual cell or cell structures harbouring the infective genome can be elucidated by ISH e.g. HBV in hepatocytes, parvovirus in cells of the lung.
Elucidation of mechanism of virus dissemination and transmission
Natural horizontal and vertical transmission routes of viruses can be studied.
For example, the presence of EBV in epithelia1 cells of the oropharynx provides a means for transmission of the virus through saliva.

68. Localisation of persistent virus infection
Examples are the persistence of JC virus in oligodendrocytes in progressive multifocal leukoencephalopathy and measles virus in neurons and glia cells in SSPE.
Link between virus agents and carcinogenesis
Etiological role of various viruses in cancers and the mechanisms of malignant transformation of cells.
The better known associations are:
EBV and nasopharyngeal carcinoma and B cell lymphomas,
HBV and hepatocellular carcinomas and
HPV and cervical carcinoma.

69. Study of cell development
ISH detection of cell-type specific RNA in cells which do not exhibit morphological differentiation can be applied to identify the cell type.
Sex determination
The Y chromosome can be detected through hybridisation.
Human gene mapping
Interphase cytogenetics
ISH can be used to detect numerical chromosomal abberrations in interphase nuclei.
Probes recognising highly repetitive sequences in chromosomes 1,7,8,9, 10, 15, 16, 17, 18, X and Y are now available.

70. FISH

71. Soon after Gall and Pardue's work, fluorescent labels quickly replaced radioactive labels in hybridization probes because of
Greater safety,
Stability, and
Ease of detection
(Rudkin & Stollar, 1977).
In fact, most current in situ hybridization is done using FISH procedures.

72. METHODOLOGY The basic steps in a FISH procedure include…
Fixation of the DNA, as either metaphase chromosomes or interphase nuclei, on a slide;
DNA is then denatured in situ, so that it becomes single stranded.
This target DNA is then hybridized to specific DNA probe sequences, which are labelled with fluorochromes
To allow for their detection.

73. PROBES ARE USUALLY DIRECTLY LABELLED INDIRECTLY LABELLED
Fluorochrome is directly attached to the probe nucleotides.
INDIRECTLY LABELLED
via incorporation of a hapten (such as biotin or digoxigenin).
Probes are then detected using a fluorescently labeled antibody (such as strepavidin and antidigoxigenin).
Probes and targets are finally visualized in situ by microscopy analysis.

74. Basic elements are a DNA probe and a target sequence.
(B) Before hybridization, DNA probe is labelled
Indirectly with a hapten
(left panel) or
Directly labelled via incorporation of a fluorophore (right panel).

75. (C) The labelled probe and the target DNA are denatured to yield single stranded DNA.
D) They are then combined, which allows the annealing of complementary DNA sequences.

76. (E) If the probe has been labelled indirectly, an extra step is required for visualization of the non-fluorescent hapten that uses
Enzymatic or
Immunological detection system.
(F) Finally, the signals are evaluated by fluorescence microscopy.

78. Whereas FISH is faster with directly labeled probes, indirect labelling offers the advantage of signal amplification by using several layers of antibodies, and it might therefore produce a signal that is brighter compared with background levels.
Currently, directly labeled probes may be labelled in…
Green (such as Spectrum Green TM or fluorescein),
Red (Spectrum Orange TM or Texas Red),
Blue (Spectrum Aqua TM), or
Gold (Spectrum Gold TM).

79. FISH can be applied to a variety of clinical specimens, providing there is DNA in the sample.
Cultured cells, such as..
Amniocytes,
Chorionic villi,
Lymphocytes,
Bone marrow aspirates, or
From solid tumors

80. CLINICAL APPLICATIONS
OF FISH

81. PRENATAL CHROMOSOME STUDIES
One of the major advantages of FISH is the ability to detect numerical abnormalities (aneuploidy) in uncultured cells from amniotic fluid or chorionic villi.
In high-risk pregnancies, including those associated with
advanced maternal age (older than 35 years),
abnormal ultrasound findings, or
abnormal maternal screening results, .

82. PRENATAL CHROMOSOME STUDIES
FISH is used as an adjunct to standard cytogenetic analysis to provide aneuploidy screening for chromosomes 13, 18, 21, as well as the X and Y chromosomes.
Aneuploidy of these chromosomes accounts for the most common abnormalities detected prenatally.
FISH technology on prenatal samples has been found to be effective, sensitive, and specific (Tepperberg et al. 2001).

83. MICRODELETION AND MICRODUPLICATION SYNDROMES
Microdeletion or contiguous gene syndromes are caused by a deletion of genetic material, which results in the loss of several genes from one chromosomal region.
Generally, these deletions are <2 Mb in size.
There are several, clinically recognized, microdeletion syndromes, for which commercial FISH probes are available.
Microduplication syndromes are caused by a gain of genetic material, often in the same regions of the chromosome in which microdeletions are observed.
The same FISH probes used for microdeletion analyses may be used for microduplication analyses; the major difference is that interphase nuclei must be
scored as well as metaphase cells.

84. FISH testing for a deletion. Two probes are usually used;
First probe (green) is a control probe It hybridises to a sequence that is not part of the deletion, so a signal is observed on each chromosome.
Second probe (red) hybridises to the sequence that may be deleted. A deletion is usually found in only one of the chromosomes in a pair, therefore the probe can bind to the intact chromosome, but is unable to bind to the deleted chromosome and only one signal is seen.

85. ACQUIRED ABNORMALITIES
FISH probes have been developed for the majority of recurrent chromosomal aberrations found in haematological malignancies.
One of the commercial suppliers of haematological FISH probes is Abbott Molecular Inc., an Abbott Laboratories Company (Des Plaines, IL).

86. SOLID TUMORS
Solid tumors are often difficult to grow in culture, and metaphase spreads can be hard to obtain and/ or analyze.
FISH is useful in detecting specific rearrangements in interphase cells of solid tumors that have diagnostic and prognostic implications.

87. NEUROBLASTOMA
Amplification and overexpression of the MYCN oncogene on chromosome 2 is seen in childhood neuroblastoma and is associated with rapid tumor progression and a poor prognosis (Ambros et al. 2009).
The MYCN FISH probe is used to detect extra copies or amplification of the gene.
Analysis is usually performed on interphase cells, from bone marrow biopsies, fresh or snap-frozen tumor, or paraffin-embedded tumor samples.

89. Fluorescence in-situ hybridization of MYCN probe to metaphase and interphase nuclei of a primary neuroblastoma with
MYCN amplification.