Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality.

Area of Science:

• Molecular Biology
• Neuroscience
• Genomics

Background:

• Spatial transcriptomics preserves gene expression and spatial context in tissues, unlike bulk or single-cell sequencing.
• Combining spatial transcriptomics with immunofluorescence staining (e.g., 10× Genomics Visium) allows linking gene expression to cellular morphology and interactions.
• Existing protocols may compromise RNA quality or limit antibody choices for combined analyses.

Purpose of the Study:

• To develop an optimized protocol for combining spatial transcriptomics with immunofluorescence staining on fresh frozen mouse brain slices.
• To investigate and improve parameters affecting both RNA integrity and antibody binding.
• To enhance the compatibility of the protocol for a broader range of antibodies.

Main Methods:

• Application of the 10× Genomics Visium platform to fresh frozen mouse brain cryo-sections.
• Systematic investigation of fixation time and buffer composition effects on RNA quality and antibody staining.
• Development and validation of a modified protocol based on experimental findings.

Main Results:

• The developed protocol successfully preserves RNA quality during immunofluorescence staining.
• The improved protocol enhances buffer compatibility, enabling the use of additional antibodies not supported by the manufacturer's protocol.
• Optimization of fixation and buffer parameters was crucial for balancing RNA integrity and antibody signal.

Conclusions:

• An improved protocol for combined spatial transcriptomics and immunofluorescence staining in mouse brain tissue has been established.
• This optimized protocol maintains high RNA quality and broadens the scope of immunofluorescence applications.
• The findings provide a foundation for developing tailored staining protocols for specific spatial biology research needs.