Multiplexed Immunofluorescence and Single-Molecule RNA Fluorescence In Situ Hybridization in Mouse Skeletal Myofibers

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• RNA fluorescence in situ hybridization (FISH) is vital for studying mRNA abundance and location.
• Existing RNA FISH methods are primarily suited for cell cultures, not complex in vivo tissues.
• Skeletal muscle presents unique challenges for single-molecule RNA detection, including tissue thickness and autofluorescence.

Purpose of the Study:

• To establish a robust method for single-molecule RNA detection in isolated adult mouse skeletal muscle myofibers.
• To enable simultaneous detection of mRNA and proteins within these specialized cells.

Main Methods:

• Isolation of adult myofibers from mouse skeletal muscle.
• Application of multiplexed RNA FISH for single mRNA molecule detection.
• Integration of immunofluorescence for simultaneous protein detection.

Main Results:

• Successful isolation of viable adult myofibers.
• Demonstration of single-molecule RNA detection within these myofibers.
• Validation of multiplexed RNA FISH and immunofluorescence for co-detection of RNA and proteins.

Conclusions:

• The presented method overcomes limitations of traditional RNA FISH in thick tissues.
• This technique provides a powerful tool for in vivo molecular analysis of skeletal muscle.
• Enables detailed spatial and quantitative analysis of gene expression in myofibers.