3D-FISH on cultured cells combined with immunostaining

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Three-dimensionally preserved nuclei (3D-FISH) combined with immunocytochemistry and 3D fluorescence microscopy is crucial for analyzing interphase nuclear functional organization.
• 3D-FISH on cultured cells is a routine technique in nuclear biology, enabling visualization of chromosome territories, subregions, genes, and RNA transcripts in their native spatial positions.
• Combining 3D-FISH with immunostaining is desirable for mapping DNA/RNA and protein targets within the same cells.

Purpose of the Study:

• To describe optimized protocols for combining 3D-FISH and immunostaining.
• To address challenges where FISH procedures can interfere with antibody staining.
• To provide reliable methods for simultaneous spatial mapping of nucleic acids and proteins in the nucleus.

Main Methods:

• Development and validation of three distinct variants of combined 3D-FISH and immunostaining protocols.
• Utilizing 3D fluorescence microscopy for high-resolution imaging.
• Careful optimization of FISH steps to minimize interference with subsequent antibody staining.

Main Results:

• Successful implementation of three combined 3D-FISH and immunostaining protocols in the laboratory over many years.
• Demonstrated ability to visualize and map both DNA/RNA and protein targets simultaneously within the 3D nuclear architecture.
• Preservation of spatial positioning of visualized targets.

Conclusions:

• The presented protocols offer robust methods for dual-target analysis in nuclear biology.
• Combined 3D-FISH and immunostaining is an effective strategy for detailed nuclear organization studies.
• These techniques advance the understanding of functional nuclear organization by integrating nucleic acid and protein localization data.