Iterative Bleaching Extends Multiplexity (IBEX) is a highly multiplexed immunohistochemistry technique that increases our capacity to gain spatial information by allowing multiple rounds of immunohistochemistry to be performed on a single tissue sample. We have adapted the IBEX method to visualise the many cell types in the zebrafish retina at once. The rich spatial detail that this method provides is useful in the study of retina development, in which there is a complex interplay between multiple cell types to make the precise neural circuits necessary for vision. Understanding how these cells come together and degenerate in disease is challenging due to our limited ability to resolve multiple cell types in the same tissue. The use of micro-conjugation kits with the IBEX method allows us to overcome the issues faced in multiplexed immunohistochemistry, allowing us to use antibodies which have been raised in the same species together in the same panel. We have successfully imaged 11 markers over three rounds of immunohistochemistry on zebrafish retina cryosections. IBEX allows many more than the usual three or four markers per tissue sample that many researchers are limited to, to be used on a single sample and it has the potential to be adapted for use in other types of tissue, depending on the biological questions being asked. In this way, we believe that the IBEX method will be a useful tool for the zebrafish research community.
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