'Iterative Bleaching Extends Multiplexity' facilitates simultaneous identification of all major retinal cell types.

Area of Science:

• Neuroscience
• Developmental Biology
• Cell Biology

Background:

• Understanding tissue multicellular composition is crucial for studying development, aging, and disease.
• Current limitations in combining multiple cellular markers hinder comprehensive cellular landscape visualization.

Purpose of the Study:

• To adapt and optimize the Iterative Bleaching Extends Multiplexity (IBEX) technique for the zebrafish retina.
• To enable simultaneous visualization of all major retinal cell types using multiplexed immunofluorescence.
• To demonstrate the broad applicability of IBEX across different species and experimental techniques.

Main Methods:

• Adaptation of Iterative Bleaching Extends Multiplexity (IBEX) for zebrafish retinal tissue.
• Optimization of fluorescent antibody micro-conjugation for sequential labeling.
• Integration of IBEX with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and whole-mount immunofluorescence (WMIF).
• Application of IBEX across multiple developmental stages.

Main Results:

• Successful visualization of all major retinal cell types in zebrafish using 11 cell-specific antibodies via IBEX.
• Demonstrated compatibility of IBEX with transgenic lines, HCR, and WMIF.
• Applied IBEX to study spatial and temporal relationships between glia and neurons during retinal development.
• Validated IBEX utility in turquoise killifish and African clawed frog.

Conclusions:

• The adapted IBEX technique significantly enhances the ability to visualize multiple cell types within a single tissue.
• IBEX provides a powerful tool for studying cellular development, spatial-temporal relationships, and disease pathology in the retina.
• This multiplexed immunofluorescence approach has broad applicability across various species, revolutionizing cellular visualization in precious tissues.