DNA-barcode-based Multiplex Immunofluorescence Imaging to Analyze FFPE Specimens from Genetically Reprogrammed Murine...

Area of Science:

• Biotechnology
• Proteomics
• Molecular Imaging

Background:

• Formalin-fixed paraffin-embedded (FFPE) samples offer advantages for tissue analysis but lack validated antibody panels for multiplex imaging.
• Spatial proteomics requires robust antibody panels for accurate visualization of tissue microenvironments.

Purpose of the Study:

• To present a protocol for developing and validating antibody panels for multiplex imaging of FFPE tissues.
• To analyze the spatial proteomics of a murine melanoma model's tumor microenvironment.
• To establish an open-source image analysis pipeline for FFPE tissue proteomics.

Main Methods:

• Development of a DNA-barcode-based multiplex imaging technique using Co-Detection-by-indEXing.
• Application of the technique to FFPE tissues from a nanoparticle-treated murine melanoma model.
• Utilization of open-source computational tools for image analysis, cell segmentation, and spatial metric quantification.

Main Results:

• Successful visualization and spatial proteomics analysis of FFPE murine melanoma tissues.
• Demonstration of an image analysis pipeline for cell phenotyping and spatial quantification.
• Establishment of a protocol for antibody panel development in FFPE samples.

Conclusions:

• The presented protocol facilitates spatial proteomics analysis in FFPE tissues, overcoming limitations of existing methods.
• This approach enables novel insights into tumor microenvironment reprogramming and spatial biology.
• The developed tools support the design of antibody panels for diverse FFPE research applications.