A Quick Immuno-FISH Protocol for Detecting RNAs, Proteins, and Chromatin Modifications

Area of Science:

• Cell Biology
• Genetics
• Molecular Biology

Background:

• Immunofluorescence and fluorescence in situ hybridization (FISH) are crucial cytogenetic techniques for visualizing cellular molecules.
• Simultaneous detection of proteins, RNA, and DNA/chromatin modifications is essential for understanding complex biological processes.

Purpose of the Study:

• To describe a rapid, sequential immunofluorescence and RNA FISH (immuno-FISH) protocol.
• To enable simultaneous visualization of proteins, chromatin modifications, and RNAs on the inactive X-chromosome (Xi).

Main Methods:

• Developed a quick sequential immuno-FISH protocol adaptable for female mouse embryonic fibroblast (MEF) and 3T3 cell lines.
• Utilized pooled oligonucleotide probes labeled with a single fluorophore for RNA FISH.
• Optimized the RNA FISH procedure to reduce incubation time from overnight to 1-2 hours without compromising sensitivity.

Main Results:

• Successfully demonstrated simultaneous detection of proteins, chromatin modifications, and RNAs on the Xi.
• Achieved a significant reduction in RNA FISH assay duration.
• Validated the protocol's effectiveness in MEF and 3T3 cell lines.

Conclusions:

• The described immuno-FISH protocol offers a time-efficient method for multi-analyte detection in cytogenetics.
• This technique facilitates the simultaneous visualization of proteins, RNAs, and chromatin modifications.
• The protocol has broad applicability for studying various molecular targets and cellular processes.