Repeated fluorescence in situ hybridization by a microwave-enhanced protocol

Area of Science:

• Pathology
• Molecular Biology
• Genomics

Background:

• Fluorescence in situ hybridization (FISH) is a powerful technique for visualizing nucleic acid sequences within cells and tissues.
• Analyzing genomic alterations in small tissue lesions often requires multiple hybridization steps, which can be challenging with traditional methods.
• Existing FISH protocols can suffer from background noise and signal degradation after multiple probe applications.

Purpose of the Study:

• To develop and validate a novel re-hybridization protocol for pathology archive sections using microwave-assisted FISH.
• To enable efficient and repeated application of multiple probes on the same tissue section.
• To facilitate the identification of genome-wide changes in small pathological lesions.

Main Methods:

• A microwave-assisted fluorescence in situ hybridization (FISH) protocol was developed for pathology archive sections.
• The protocol involves stripping previously hybridized probes using hydrochloric acid (HCl).
• Intermittent microwave irradiation was used to re-hybridize with new probes, generating clear signals.

Main Results:

• The novel protocol successfully generated clear FISH signals with minimal background noise.
• Repeated stripping and re-hybridization with numerous bacterial artificial chromosome (BAC)-derived probes were demonstrated.
• The method proved effective for identifying genome-wide changes within small lesions on pathology sections.

Conclusions:

• The described microwave-assisted re-hybridization protocol offers a robust method for analyzing genomic alterations in pathology samples.
• This technique allows for efficient multi-probe analysis on archival tissue sections, overcoming limitations of traditional FISH.
• The protocol has significant potential for detailed molecular profiling of small lesions, aiding in diagnosis and research.