The new histologic-based FISH method involves steps similar to those found in the standard cytogenetics method. Deparaffinization and rehydration are performed in the same manner for both methods. Pre-treatment and digestion are also performed for both methods, but they are significantly different. Probes are applied and hybridization is performed similarly in both methods. On the second day, a stringent wash is performed. This ensures removal of unbound and unspecifically bound probe before dehydration with ethanol and DAPI staining in both methods.
Although the standard FISH method and the new histologic-based FISH method produce significantly comparable results, the differences in methodologies are considerable.
| Standard FISH | New Histologic-Based FISH |
- Requires that slides be heated overnight at 56° C for a minimum of 4 hours before testing is begun
| - Allows testing after microtomy completion with NO baking time
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- Uses glass coplin jars which limit the number of slides per run to nine
- Slides must be handled individually throughout the procedure
| - A container and rack is used which allows twenty-four slides per run.
- Slides are carried through the majority of the procedure as a racked group
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- Requires a heated enzymatic digestion with digestion time varying between tissue type and size of sample
| - Employs a room temperature ready-to-use pepsin
- All specimens are digested for 10 minutes initially
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- Many probes used require denaturation that must be carefully timed with slide denaturation
| - Allows the incorporation of a co-denaturation procedure
- The probe and specimen slide are denatured at the same time using a hybridizer
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