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

Multiplexed immunofluorescence (IF) can be achieved using different commercially available platforms, often making use of conjugated antibodies detected in iterative cycles. A growing portfolio of pre-conjugated antibodies is offered by the providers, as well as the possibility for in-house conjugation. For many conjugation methods and kits, there are limitations in which antibodies can be used, and conjugation results are sometimes irreproducible. The conjugation process can limit or slow down the progress of studies requiring conjugation of essential markers needed for a given project. Here, we demonstrate a protocol combining manual indirect immunofluorescence (IF) of primary antibodies, followed by antibody elution and staining with multiplexed panels of commercially pre-conjugated antibodies on the PhenoCycler platform. We present detailed protocols for applying the workflow on fresh frozen and formalin fixed paraffin embedded tissue sections. We also provide a ready to use workflow for coregistration of the images and demonstrate this for two examples.