Zucker, A.; Nguyen, C.; Brondsted, F.; Jones, J. A.; Malankar, G. S.; Ekstrom, T. J.; Rounds, C.; Ravi, D.; Goodyear, S. M.; Kardosh, A.; Wong, M. H.; Wang, L. G.; Gibbs, S. L.

Advances in spatial proteomics through the development of multiplexed immunostaining platforms have facilitated analyses with increasing cellular and molecular granularity. However, currently available approaches are limited by harsh conditions for signal removal, restricting the number of antigens that can be probed in a single specimen without significant alterations to sample quality and structure. Here we present an approach for direct labeling of primary antibodies with fluorophores using a photocleavable linker (PCL) with a polyethylene glycol spacer (PEG) to enable cyclic immunofluorescence (cyCIF) with gentle signal removal conditions. Our innovative approach uses directly labeled primary antibodies to enhance staining specificity and cyclic immunostaining efficiency, while minimizing nonspecific background signal. Additionally, through integration of the PCL, this approach facilitates gentle cleavage of antibody conjugated fluorophore, preserving sample integrity over multiple rounds of staining. Direct PEG-PCL antibody labeling will promote greater multiplexing by minimizing specimen damage and allow for quantitative analyses of cyCIF spatial data. We demonstrate that cyCIF with PEG-PCL conjugated antibodies can be applied across a variety of cancer subtypes to identify and characterize rare neoplastic cell populations in both tumor tissue and fragile peripheral blood specimens.