During the protease digestion step, timing and temperature are critical. Add the protease to the solution in advance for proper mixing and then verify that the protease solution returns to 37°C before use. Immerse slides in protease solution at 37°C for a specified digestion time (refer to the comment below). Follow the digestion time precisely, then remove the slides promptly and immerse them into the wash buffer for five minutes. Immerse slides in a second Coplin jar of wash buffer for five minutes. Slides must then be dried on a 45–50°C slide warmer for 2–5 minutes, or they may be air dried for approximately 15 minutes.
COMMENT: Digestion is a critical step in the FISH assay and must be carefully controlled. Each tissue type can vary in the strength and time of digestion needed for optimal probe binding. Breast tissue has more connective tissue than liver tissue, so it will require a longer period for digestion than liver tissue. Generally, 18–23 minutes is a good range for digestion time. The time must be determined by the laboratory testing related to the fixation time used in that institution.
Clinical laboratories commonly receive requests to confirm metastatic breast cancer lesions. When determining how much time is necessary to digest the tissue, it is important to consider the tissue to which the breast cancer has metastasized. In general, areas with a lot of connective tissue require more digestion than "softer" tissues, such as the liver.
It is important to note that bone core biopsies that have been decalcified are not amenable to FISH testing. The strong acid used in bone decalcification is detrimental to the DNA in the cells.
