# Protocol for applying expansion microscopy to the study of mammalian neuromuscular junctions

## TL;DR

This paper provides a detailed protocol for using expansion microscopy to study neuromuscular junctions in human and mouse muscle tissue at super-resolution.

## Contribution

A step-by-step protocol for applying 4× expansion microscopy to mammalian neuromuscular junctions is presented.

## Key findings

- The protocol enables super-resolution imaging of NMJ features at the nanoscale.
- It includes methods for gel embedding, digestion, and expansion of thick muscular samples.
- The technique is applicable to both human and mouse muscle preparations.

## Abstract

Expansion microscopy (ExM) is a tissue-swelling technique that enables super-resolution imaging through a specialized preparation process that physically expands stained biomolecules within a fixed sample. Here, we detail a protocol to apply the 4× ExM technique to neuromuscular junctions (NMJs) from both human and mouse muscle preparations. We describe steps for muscle fixation, microdissection, staining, gelation, and digestion. We then detail procedures for expansion, mounting, imaging, analysis, and quantification. This protocol can be used to reveal nanoscale anatomical NMJ features.

For complete details on the use and execution of this protocol, please refer to Ramadan et al.1

•Expansion microscopy of mammalian muscle: dissection, fixation, and NMJ labeling•Instructions for gel embedding, digestion, and gel expansion for thick muscular samples•Guidance on imaging and analyzing expanded NMJs at super-resolution scale

Expansion microscopy of mammalian muscle: dissection, fixation, and NMJ labeling

Instructions for gel embedding, digestion, and gel expansion for thick muscular samples

Guidance on imaging and analyzing expanded NMJs at super-resolution scale

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Expansion microscopy (ExM) is a tissue-swelling technique that enables super-resolution imaging through a specialized preparation process that physically expands stained biomolecules within a fixed sample. Here, we detail a protocol to apply the 4× ExM technique to neuromuscular junctions (NMJs) from both human and mouse muscle preparations. We describe steps for muscle fixation, microdissection, staining, gelation, and digestion. We then detail procedures for expansion, mounting, imaging, analysis, and quantification. This protocol can be used to reveal nanoscale anatomical NMJ features.