Spatial proteomic mapping of the human and mouse retina using IBEX

Purpose To generate a comparative spatial proteomic atlas of the human and mouse retina using a refined highly multiplexed immunohistochemistry technique called Iterative Bleaching Extends Multiplexity (IBEX). Methods We refined the IBEX workflow by integrating an antibody removal option alongside chemical bleaching. This dual strategy enabled removal of the entire antibody complex allowing for the flexible use of antibodies from the same host species across iterative cycles. Spatial proteomic data were cross-correlated to existing scRNAseq datasets. We also coupled this workflow with super-resolution imaging via deconvolution and applied it to the retina of healthy human, mouse and the Crb1 rd8 mouse model. Results We successfully imaged over 25 protein markers on human and mouse tissue sections, to generate spatial atlases of the major retinal cell populations. Cross-species protein expression was compared to scRNAseq datasets to identify protein and transcript disparities. Super-resolution IBEX delineated the ultrastructural features of the outer limiting membrane (OLM), identifying CD44 as a core structural component tightly co-localised with a highly organised F-actin belt within Müller glial endfeet. Using the Crb1 rd8 mouse model, disruption of this complex was spatially associated with rosette formation and OLM structural failure. Conclusion Spatial proteomic atlases of the human and mouse retina were generated and correlated to transcriptional data, revealing insights into the arrangement of major retinal cell populations and OLM structure.