In Situ Hybridization Support—Getting Started

Chromogenic in situ hybridization, or CISH, allows for detection of gene amplification, chromosome translocations, and chromosome number. CISH uses conventional peroxidase- or alkaline phosphatase-catalyzed reactions to stain 4-5 μm thick formalin-fixed, paraffin-embedded (FFPE) tissue sections. Frozen sections and cytological specimens can also be stained. The results are visualized under a conventional light microscope. CISH depends upon the ability of labeled DNA and RNA probes to hybridize (bind) in situ to specific sequences of complementary DNA or RNA in the tissue sample. CISH with DNA probes is sometimes referred to as DISH, while CISH with RNA probes is called RISH. Probe hybridization results are then imaged within the context of the surrounding tissue morphology. This means that pathologists can view tissue morphology and gene aberrations simultaneously.

Both techniques rely on the efficiency and specificity of nucleic acid hybridization to locate specific DNA and RNA sequences in cells and tissues. FISH, or fluorescence in situ hybridization, relies on DNA or RNA probes directly labeled with fluorophores or with biotin. When biotin labels are used, detection is achieved with fluorophore-labeled biotin binding proteins like streptavidin. Imaging in FISH requires an epifluorescence microscope equipped with the appropriate filters for visualizing the light emitted by the fluorophores used. Typically, fluorescence microscopes need to be equipped with a CCD camera and the results are usually processed with image analysis software. One distinct advantage of FISH is that multicolor staining (multiplexing) is possible.

CISH relies on DNA and RNA probes labeled with non-fluorescent haptens like biotin and digoxigenin. These haptenylated probes are detected in a second step using streptavidin or anti-hapten antibodies conjugated to enzymes like HRP and alkaline phosphatase. Chromogenic substrates for these enzymes are used for localizing the target nucleic acids in the sections. Such chromogens as DAB, AEC, NBT/BCIP, and Fast Red yield insoluble, colored precipitates which stain the nucleic acid targets. 

An ordinary light microscope can be used to image CISH results, and unlike FISH, histological evaluation of the tissues is possible simultaneously if the slides are counterstained appropriately. Although multicolor CISH has been performed, it is much more difficult to do successfully, compared to FISH. However, unlike FISH, slides stained with CISH can be archived like any other pathology slides.

Please view the comparison table below:

Optimum imaging of FISH results is usually achieved at a magnification of 100X. CISH results are typically resolved at a magnification of 40X.

We sell 2 types of FISH kits that enable you to assay multiple targets and visualize co-localized signals in a single specimen. Using spectrally distinct fluorophore labels for each hybridization probe, this approach gives you the power to resolve several genetic elements or multiple gene expression patterns through multicolor visual display. We offer FISH Tag™ detection kits for routine analysis, and tyramide-based TSA™ kits which permit signal amplification for detection of very rare or low-abundance targets.

Tissues should be fixed in neutral buffered formalin for at least 12-24 hours prior to paraffin embedding. Tissue sections should be 4-5 μm thick and mounted on HistoGrip™ reagent (Cat. No. 008050)-treated glass microscope slides, SuperFrost Plus, or Fisherbrand Superfrost slides. The latter have a permanent positive charge on their surfaces, which helps tissue sections and cells to adhere electrostatically to them. Thus, in most cases, there is no need to apply any special tissue adhesives or other coatings to the slides before using them. 

However, other types of glass slides are also used for IHC, CISH, and FISH, although they may need to be treated to promote tissue adherence. Commonly used methods include coating slides with various reagents which help the tissues stick to the glass. These materials include poly-L-lysine, chrome alum-gelatin, Mayer's albumin, starch paste, collagen, and 3-aminopropyltriethoxysilane (APES or TES). Our slide coating reagent called HistoGrip™ reagent (Cat. No. 008050) is strongly recommended. HistoGrip™ reagent creates a strong bond between the tissue and the slide surface. Enzyme digestion and other antigen retrieval procedures, and even the temperatures used in CISH and FISH will not remove tissues attached using HistoGrip™ concentrate. Our testing showed that a HistoGrip™ concentrate coating results in better tissue adherence than one composed of poly-L-lysine. No special de-paraffinization and re-hydration steps are necessary prior to CISH or FISH, although we strongly recommend that you use fresh solvents to prevent paraffin and xylene carry-over.

One step of central importance in CISH is the incubation temperature during the heat treatment step. This represents the first antigen retrieval step in the CISH protocol. It is critical have the specimen be heated 98°C for 15 minutes in the pretreatment buffer included in the Spot-Light Tissue Pretreatment Kit (Cat. No. 008401) that we recommend. This can be accomplished in one of the following ways.

1) Microwave Oven: Place the slides in a plastic Coplin jar containing the pretreatment buffer and cap it loosely. Place a temperature probe in a separate jar containing water or buffer, but without a cap. Set the temperature to 93°C. Set the timer for 15 minutes when the temperature reaches 93°C. Note that the temperature in the jar with the cap should be ~98°C. Slides can be transferred to deionized water immediately afterwards. 

2) Pressure cooker: Once the water is boiling inside the cooker, set the timer for 10 minutes and apply the steam nozzle weight. Open the pressure cooker when the internal pressure has dropped to 0, at which point the temperature inside will be around 95°C. 

3) Hot plate: Heat the pretreatment buffer in a beaker. After the buffer temperature reaches above 98°C, put the slides in and heat them for 15 minutes. To prevent the buffer from evaporating, cover the beaker loosely with a glass cover or aluminum foil. 

4) Steamer: Make sure the pretreatment buffer temperature reaches 98-100°C before putting the slides in the container. Then, steam them for 20 minutes. 

It is important to note that the incubation time starts after the buffer is pre-heated to the required temperatures, not while it is heating up. 

The pepsin digestion step represents the second phase of antigen retrieval. This step can be performed at room temperature or at 37°C. For the former, let the pepsin solution come to room temperature first. If you want to perform digestion at 37°C, you should pre-warm the pepsin to 37°C before applying it to your sections. Depending on the tissue type and fixation method used, different incubation times may be required. For example, we suggest a 10-minute pepsin digestion at room temperature or 3 min at 37°C for human breast tissue sections. 

We have observed that over-digestion can weaken or eliminate the CISH signal and prevent counterstaining of cell nuclei. Under-digestion may also decrease or eliminate the CISH signal, but you will still be able to counterstain the nuclei.

For denaturation, the lowest incubation temperature and time we suggest is 92°C for 2 min. For best results, we recommend using 94-95°C for 5 min. 

Our recommended procedure for applying the probe to the slides is as follows. Depending on the size of the tissue section, a starting volume of 15 µL of the probe can be applied either to the coverslip or straight on to the tissue section with a pipette. The edges of the coverslip are then sealed to the slide with rubber cement. If you decide not to coverslip the slides, it is important to perform subsequent incubations at 37°C in a humidified chamber. 

For hybridization, 12 hours at 37°C may be sufficient, but we suggest overnight incubation for best results. In fact, as long as the specimen does not dry out, the hybridization can be extended to 72 hours, if desired. We suggest that you securely seal the cover slips to your slides by applying rubber cement to the edges to form a tight seal

No, CISH and FISH can be performed with cytological specimens, but not with frozen sections.

For CISH with cell preparations, the pretreatment step is different than for FFPE sections. With cells, we recommend omitting the heat treatment step, but using the pepsin enzyme treatment for antigen retrieval. The remainder of the protocol for CISH with FFPE is the same. For cell or metaphase chromosome samples, we recommend the following modified CISH protocol:

1) Immerse the slides in 2x SSC buffer for 60 minutes at 37°C. 

2) (Optional) Pretreat cells with SPoT-Light™ Cell Pretreatment Reagent (Cat. No. 00-8402) for 5 min at 37°C. Variations in incubation time (2-10 min) may be required, depending on the cell type and how the slides are made. Remember that over-digestion with pepsin will cause loss of nuclei and chromosome structure, while inadequate digestion may result in loss of signal. 

3) Wash the slides three times in distilled water at room temperature for 2 minutes.

4) (Optional) Post-fix the cells by Immersing the slides in 10% buffered formalin for 1 minute at room temperature. 

5) Wash the slides three times in distilled water at room temperature for 2 minutes each. 

6) Dehydrate slides in 70%, 85%, 95%, and 100% ethanol for 2 min each, and then air-dry them for 20 minutes at room temperature. 

7) The slides are now ready to enter the CISH staining protocol. Proceed to the denaturation and hybridization steps. You should then add the probe, hybridize overnight, and perform the detection as usual.