Immunolabel-First-Expand-Later Expansion Microscopy Approach Using Stable STED Dyes

Area of Science:

• Cell Biology
• Microscopy Techniques
• Molecular Imaging

Background:

• Expansion microscopy (ExM) enables nanoscale imaging of cellular structures like centrioles, centrosomes, and cilia.
• Current ExM protocols often face challenges with antibody binding to proteins after sample expansion and denaturation.
• Precise localization of centrosomal and ciliary proteins is crucial for understanding cell function.

Purpose of the Study:

• To develop and validate an expansion microscopy protocol using stable STED dyes for improved protein detection.
• To overcome limitations in primary antibody binding to proteins post-denaturation in ExM.
• To enable robust nanoscale imaging of centriolar and ciliary proteins using an 'immunolabel-first-expand-later' approach.

Main Methods:

• Utilized two stable STED dyes compatible with expansion microscopy protocols.
• Implemented an 'immunolabel-first-expand-later' strategy, performing immunolabeling before sample expansion.
• Validated dye stability through crosslinking, polymerization, and denaturation steps of the ExM protocol.

Main Results:

• Demonstrated the stability of STED dyes throughout the expansion microscopy workflow.
• Achieved robust detection of immunolabeled proteins using both conventional and STED microscopy after expansion.
• Successfully analyzed the localization of the centriole appendage protein Cep164 and the ciliary protein ARL13B.

Conclusions:

• The developed ExM protocol with stable STED dyes offers a reliable method for nanoscale protein localization.
• This approach effectively addresses the challenge of antibody accessibility after protein denaturation in ExM.
• The method provides a valuable tool for advancing the study of centrioles, centrosomes, and cilia.