Method of the Month: Chromogenic In Situ Hybridization
For this month, our method will be chromogenic in situ hybridization, or CISH. It is a tool used by biologists to visualize the presence of specific DNA regions within cells and has important applications in the medical field.
Chromogenic in situ hybridization (CISH) was developed just over two decades ago by a group of geneticists searching for a better way to detect HER-2/neu oncogene amplification in breast cancer patients. This gene of interest, HER-2/neu, codes for a protein involved in normal cell growth. However, in about 15%-20% of breast tumors, this gene is amplified, or overexpressed. An existing method similar to CISH was not very practical for use in diagnostic laboratories because it required expensive, high-resolution fluorescence microscopes and cameras. This technique, fluorescence in situ hybridization (FISH), was featured in an earlier method of the month article, which can be read here.
CISH consists of five basic steps: probe design, tissue preparation, probe hybridization, pre-detection treatment, and probe detection. The first step, probe design, involves creating a sequence of DNA approximately 20 nucleotides long that will bind to a complementary sequence in the target gene. Once a probe is created, copies of it need to be made through an amplification process such as a polymerase chain reaction. These probes are then labeled either with biotin (Vitamin B7) or digoxigenin (a steroid isolated from the foxglove plant).
In tissue preparation, a piece of tissue from a biological sample, such as a tumor, is affixed to a microscope slide using paraffin. In order to remove excess wax from the glass, the slide must be washed and heated several times. Finally, the enzyme pepsin will be applied to digest proteins in the sample, making the target DNA more readily accessible.
The step of probe hybridization consists of binding the probe to its targets within the tissue sample. Approximately 10–20 µL of probe is added to the slide, and a coverslip is then sealed overtop of it. For the probe to be able to bind to its complementary sequence, the sample DNA must be denatured, or separated into single strands. This is accomplished by heating the slide to 97°C for five to ten minutes.
To ensure that only the target gene will be bound to a probe, a pre-detection treatment is applied. This involves adding a substance to the slide that will bind to all non-specific binding sites, leaving only the specific binding site of the probe open. The treatment used in this step is specific to the probe’s label. Bovine serum albumin (a protein isolated from cow blood plasma) is used if the probe was labeled with biotin, while an anti-digoxigenin fluorescein primary antibody will be used if the probe was labeled with digoxigenin.
In the final step of probe detection, a second substance will then be added to label these nonspecific sites. This is again specific to the probe’s original label, with the protein streptavidin binding to bovine serum albumin, and anti-fluorescein secondary antibody binding to the fluorescein of the primary antibody. Finally, the slide is placed under a bright-field microscope so that the presence or absence of the target gene can be visualized. In order to make the results more visible, a counterstain may be added.
To see a picture of what the final step of this method looks like under a microscope, click here!
