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 specified digestion time (refer to comment below). Follow the digestion time EXACTLY, then remove the slides promptly and immerse into 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 one that has to be carefully controlled. Each type of tissue can vary in the strength and time of digestion needed for optimal probe binding. Breast tissue has more connective tissue than liver. Breast tissue will therefore require a longer time period for digestion than liver tissue. In general, 18-23 minutes is a good range for digestion time. The time must be determined by the laboratory performing the testing, as it is related to the fixation time used in that institution.
In any clinical laboratory, it is common to have requests for metastic breast cancer lesions. It is important to consider the tissue to which the breast cancer has metastasized when considering how much time is necessary to digest the tissue. In general, areas that are connective tissue rich require more digestion than "softer" tissues, such as 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 decalcification of bone is detrimental to the DNA in the cells.