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What is ISH?
ISH stands for In Situ Hybridization. ISH is a technique for detecting specific nucleic acid sequences in cells and tissues by hybridization of a labeled probe to the specific nucleic acid sequence, and the subsequent detection of the hybridized probe. With ISH, it is possible to quantify a specific mRNA and simultaneously determine its cellular location. This powerful technique allows the temporal and spatial patterns of gene expression to be determined. ISH is also used for the detection of intracellular pathogens with a very high degree of sensitivity.
What are the advantages of the ISH Detection system?
The ISH Detection system is a complete non-isotopic detection system for all ISH applications containing all required ready-to-use reagents. Advantages include:
- Very high specificity inherent to: DNA-DNA , RNA-RNA, antibody-hapten and biotin-streptavidin interactions
- Matched detection reagents and protocols for maximal signal-to-noise ratios
- Very high sensitivity due to serial amplification
- Easy-to-use and final results in less than a day
What are the principles for the ISH fluorescein or digoxigenin-labeled probes?
The ISH Detection System for fluorescein or digoxigenin-labeled probes is designed to detect nucleic acid targets in tissues and cells by the hybridization of a fluorescein/digoxigenin-labeled probe to a specific nucleic acid sequence within the cell. This is followed by the detection of the hybridized probe by a colorimetric, chemiluminescent, or immunofluorescent reaction.
What are the steps of the protocol for Link-Label or Polymer-HRP ISH Detection system?
The steps of the process are as follows:
- Tissues or cell preparations are frozen or fixed, sectioned and attached to slides.
- The sections are de-waxed, if paraffin-embedded, treated with proteinase, and hybridized with the labeled probe
- The bound probe is localized by addition of an anti-fluorescein/digoxigenin antibody
- The bound antibody is detected in the following ways depending on the type of detection system.
- With Link-Label ISH Detection system: The signal is amplified by addition of a biotinylated secondary antibody fragment, followed by streptavidin-enzyme conjugate.
- With Polymer-HRP system: SuperBlock is applied and after incubation is followed by Poly-HRP Reagent which is again followed by incubation.
- A suitable enzyme substrate is then added and converted by the enzyme to a colored, luminescent or fluorescent end-product
- The slides are then counterstained, mounted and examined
Can the probe be detected directly under a fluorescence microscope?
No, BioGenex probes cannot be visualized directly. A detection system has to follow the reaction of the probe with the target present on the tissue or cells.
Can one use BioGenex ISH kits to detect mRNA probes?
Yes. BioGenex ISH kits allow the customer to detect either type of probes. The kits come with an RNAse Block for those working under RNAse-free conditions. In addition, all the contents of the kit are RNAse free.
What controls should be performed for ISH assays?
For initial validation of ISH, a minimum set of controls should be run with the probe and tissue to be tested.
- The initial negative control is suggested to be the hybridization buffer only (BS-1510-06) without any probe
- Other negative controls are the omission of anti-biotin or anti-fluorescein antibody only, omission of the secondary antibody only and omission of the streptavidin-enzyme conjugate only
- Negative controls should show no staining if the reaction is specific to the probe
- A positive control is performed with a known probe that binds to a ubiquitous sequence and/or with the test probe in cells that are known to contain the sequence
- Positive control staining should be seen at the required intensity and in the expected places
Why does my negative control show strong signal?
- The signal is due to non-specific cross-reactivity of detection reagents
- The tissue may contain substances that bind one of the reagents: anti-probe antibody, secondary antibody, biotin, and streptavidin-enzyme conjugate
- In some cases, the intrinsic enzyme may have interfering activity.
Why does my positive control have no signal?
In most cases, either the mRNA is degraded or the tissue is not adequately permeabilized. Too stringent hybridization or wash conditions may also weaken the signal.
What references could you provide if I want to learn more about ISH?
Larochelle R (1997) Detectiojn of porcine reproductive and respiratory syndrome virus in paraffin-embeded tissues: comparison of immunohistochemistry and in situ hybridization.
J Virol Methods 63(1-2), 227-235.
Pohle T (1996) Exprression of type I and IV collagen mRNAs in healing gastric ulcers - a comparative analysis using isotopic and non-radioactive in situ hybridization.
Histochem Cell Biol 106(4), 413-418.
Berndt A (1996) Reduced formamide content and hybridization temperature results in increased non-radioactive mRNA in situ hybridization signals. Acta Histochem 98(1), 79-87.
Besancon R (1995) Rapid in siyu hybridization using digoxigenin probe and microwave oven. Cell Mol. Biol (Noisy-le-grand) 41(7), 975-977.
Morvan PY (19995) Distinct pattern of IL-2 and IFN-gamma gene expression in CD4 and CD8 T cells: cytofluorometric analysis at a single cell level using non-radioactive probes.
Cell Mol Biol (Noisy-le-grand) 41(7), 945-957.
Macville MV (1995) Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection contrast microscopy and transmission electron microscopy. J histochem Cytochem 43(7), 665-674.
Guiot Y (1995) The effects of varying key steps in the non-radioactive in situ hybridization protocol: a quantitative study. Histochem J 27(1), 60-68.
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