Making Multiplexed Imaging Flexible: Combining Essential Markers With Established Antibody Panels

Area of Science:

• Biomedical research
• Immunohistochemistry
• Molecular pathology

Background:

• Multiplexed immunofluorescence (IF) enables simultaneous detection of multiple biomarkers in tissue samples.
• Current methods often rely on iterative cycles of antibody conjugation, which can be time-consuming and yield irreproducible results.
• Limitations exist in antibody compatibility and reproducibility with existing in-house and commercial conjugation kits.

Purpose of the Study:

• To develop and validate a novel protocol for multiplexed immunofluorescence (IF) on the PhenoCycler platform.
• To overcome the challenges associated with antibody conjugation, including limitations in antibody choice and reproducibility.
• To provide a robust and adaptable workflow for multiplexed biomarker analysis in various tissue types.

Main Methods:

• A protocol combining manual indirect immunofluorescence (IF) of primary antibodies followed by antibody elution.
• Subsequent staining with multiplexed panels of commercially pre-conjugated antibodies on the PhenoCycler platform.
• Detailed protocols for fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections, including image coregistration.

Main Results:

• Demonstration of a reproducible and efficient multiplexed IF workflow.
• Successful application of the protocol on both fresh frozen and FFPE tissue sections.
• Validation of the workflow through coregistration of images and presentation of two case examples.

Conclusions:

• The presented protocol offers a viable alternative to traditional antibody conjugation methods for multiplexed IF.
• This approach enhances flexibility and reproducibility in biomarker studies using the PhenoCycler system.
• The workflow facilitates advanced spatial biology research by enabling efficient multiplexed biomarker detection.