High Precision Antibody-Free Microtubule Labeling for Expansion Microscopy.

Expansion microscopy (ExM) enables nanoscale imaging of biological structures using standard fluorescence microscopes. Accurate labeling of cytoskeletal filaments, such as microtubules, remains challenging due to structural distortion and labeling inaccuracy during sample preparation. This protocol describes an optimized method combining detergent extraction and NHS-ester labeling for high-precision visualization of microtubules in expanded samples. Cytoplasmic components and membranes are selectively removed, preserving the ultrastructure of the microtubule network. Microtubules are digested into peptides during expansion and subsequently labeled at their N-termini using NHS-ester dyes, eliminating the need for antibodies. Effective fluorophore displacement of ~1 nm or lower is achieved, depending on the applied expansion factor. The protocol is compatible with both in vitro and cellular samples and can be integrated into a wide range of ExM workflows. Labeled microtubules can serve as internal reference standards for correcting expansion factors in ExM datasets. Key features • Employs detergent extraction with accessible commercial reagents to isolate cytoskeletal structures and reduce background from membranes and cytoplasmic proteins in fluorescence microscopy. • Avoiding excessive aldehyde fixation preserves amines required for gel polymer integration while NHS-ester labeling of tubulin amines reduces linkage error, enabling accurate molecular localization. • Compatible with post-expansion workflows; labels newly generated peptide N-termini after digestion, enabling high-resolution fluorescence imaging with minimal linkage error and high signal-to-noise ratio (SNR). • Suitable for integration into diverse ExM protocols and useful as a reference standard for expansion factor correction.