Advancing Multiplex Imaging: A Sequential Antigen Retrieval Protocol to Enhance Antibody Compatibility and Detection...

Antigen retrieval remains a major challenge in multiplex tissue imaging, as the conflicting requirements of antibodies often limit the use of certain antibodies within a panel. Various protocols for epitope retrieval were tested to develop a standardized method for optimizing signal detection for a broad range of markers intended for multiplex imaging, such as imaging mass cytometry (IMC).

Enzymatic, intermediate-pH, and heat-induced epitope retrieval (HIER) on several types of tissue were set to determine their impact on antibody compatibility and signal detection.

Our findings demonstrate that enzymatic and intermediate pH recovery methods are ineffective for most tested antibodies. Conversely, a sequential HIER approach, using citrate buffer at pH6 followed by EDTA buffer at pH9, significantly enhances the detection of diverse antigens. This two-step protocol improved staining intensity in both immunohistochemistry (IHC) and IMC, increasing the signal for 19 out of 23 antibodies tested in IMC assays across multiple tissue types.

These results establish a robust and accessible strategy to overcome antibody compatibility, which remains a major bottleneck in IMC. The proposed sequential antigen retrieval protocol provides a practical method to expand antibody panels and improve signal detection for multiplex tissue analysis.