Protocol for quantifying muscle fiber size, number, and central nucleation of mouse skeletal muscle cross-sections using Myotally software

Pieter Both; Soochi Kim; Jengmin Kang; Marina Arjona; Daniel I Benjamin; Christopher W Nutter; Armon Goshayeshi; Thomas A Rando

doi:10.1016/j.xpro.2024.103555

Protocol for quantifying muscle fiber size, number, and central nucleation of mouse skeletal muscle cross-sections using Myotally software

STAR Protoc. 2025 Jan 10;6(1):103555. doi: 10.1016/j.xpro.2024.103555. Online ahead of print.

Authors

Pieter Both¹, Soochi Kim², Jengmin Kang³, Marina Arjona³, Daniel I Benjamin³, Christopher W Nutter³, Armon Goshayeshi³, Thomas A Rando⁴

Affiliations

¹ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA; Stem Cell Biology and Regenerative Medicine Graduate Program, Stanford University School of Medicine, Stanford, CA 94305, USA.
² Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong 30019, Republic of Korea.
³ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁴ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA; Neurology Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA. Electronic address: trando@mednet.ucla.edu.

PMID: 39799576
DOI: 10.1016/j.xpro.2024.103555

Abstract

Here, we present a protocol for using Myotally, a user-friendly software for fast, automated quantification of muscle fiber size, number, and central nucleation from immunofluorescent stains of mouse skeletal muscle cross-sections. We describe steps for installing the software, preparing compatible images, finding the file path, and selecting key parameters like image quality and size limits. We also detail optional features, such as measuring mean fluorescence. By automating these traditionally labor-intensive processes, Myotally improves research efficiency and data consistency.

Keywords: Cell Biology; Computer sciences; Microscopy.