ABSTRACT
The vertebrate retina is a complex multicellular tissue made up of distinct neuron types and glia, arranged in a stereotypic layered organisation to facilitate vision. Understanding how these cell types come together to form precise circuits during development requires the ability to simultaneously discriminate between multiple cell types and their spatial position in the same tissue. Currently, we have a limited capacity to resolve all constitutive cell types and their relationships to one another, due to our limited ability to combine multiple cellular markers or antibodies. To extend this capacity, we have adapted a highly multiplexed immunohistochemistry technique known as Iterative Bleaching Extends Multiplexity (IBEX) and applied it to the development of the zebrafish retina. IBEX allows for multiple rounds of cellular labelling to be performed before integration of the imaging data with open-source software, thereby facilitating visualisation of multiple markers on the same tissue section for analysis. We have optimised fluorescent micro-conjugation of known antibody markers with sequential imaging and bleaching to label the complete zebrafish retina with up to 11 cell-specific antibodies. We have further adapted the IBEX technique to be compatible with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and wholemount immunohistochemistry. Finally, we have applied IBEX at multiple stages of retinal development to study the spatial and temporal relationships between glia cells and neurons during development, demonstrating progressive specialisation of retinal cell types consistent with previously described histogenesis. Ultimately, these techniques can be applied to any tissue in zebrafish to rapidly explore multiple cell types and biological processes in single tissue samples.
Competing Interest Statement
The authors have declared no competing interest.