Need help for antibody specificity in multiplexed imaging platforms

Antibody Based Technologies Monoclonal/Polyclonal Abs
1

Hi

What are the best practices for validating antibody specificity in multiplexed imaging platforms like CODEX or MIBI?

Thanks

2

Laura

Validating antibody specificity is essential for accurate results in multiplexed imaging platforms like CODEX and MIBI. Best practices include orthogonal validation using RNA data or spatial transcriptomics to confirm expression patterns. Use genetic knockout or knockdown models to ensure signal loss in the absence of the target protein. Validate antibodies with conventional IHC or IF and optimize concentrations through titration in single-plex before adding to the multiplex panel. Always confirm that conjugation (e.g., DNA barcoding or metal tagging) does not compromise antibody performance.

You can include internal biological controls and ensure marker co-expression follows known biology. Avoid cross-reactivity by checking for signal bleed-through, especially in high-plex settings.

You can also use pre-validated antibodies when available and include negative controls to detect background or autofluorescence.

Together, these steps minimize artifacts and increase the reliability of multiplexed spatial data in complex tissues.

Hope this helps.

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