Methods for In situ Quantification of Mitochondrial Morphology in Muscle and Terminal Schwann Cells of Mice

Summary

Local injections of a mitochondrial dye label terminal Schwann cell mitochondria in vivo for confocal microscopy. This method utilizes proof of concept demonstrated in skeletal muscle tissue from mice with healthy or diseased muscle to provide high-resolution mitochondrial visualization.

Abstract

Mitochondrial network morphology is widely used as an indicator of cellular health; however, quantifying mitochondrial architecture within intact tissues remains technically challenging. Terminal Schwann cells (tSCs), which are essential for the maintenance and regeneration of neuromuscular junctions, are particularly difficult to analyze in situ due to their anatomical location and sensitivity to tissue disruption. This protocol describes a reproducible approach for labeling and quantifying three–dimensional mitochondrial network morphology in whole-mount skeletal muscle and in tSCs in mice using standard confocal microscopy. The method employs in vivo delivery of a membrane potential–sensitive mitochondrial dye followed by rapid tissue processing and high-resolution confocal imaging. Image stacks are analyzed to quantify mitochondrial network connectivity, area, and fragmentation. The protocol is first validated in dystrophic and healthy skeletal muscle to confirm expected differences in mitochondrial morphology and is subsequently adapted to visualize and quantify mitochondrial networks in tSCs identified using S100β reporter mice. This approach enables the analysis of mitochondrial morphology within intact neuromuscular tissues without requiring transgenic mitochondrial reporters or specialized imaging platforms. The protocol requires only equipment commonly available in university core facilities and can be adapted to other thin or superficially accessible tissues.

Introduction

Mitochondria are remarkably plastic organelles that act as the lungs of the cell, consuming oxygen during cellular respiration to support adenosine triphosphate (ATP) production in response to metabolic demand¹. While once thought of as discrete organelles, it is now understood that mitochondria are highly integrated^2,3,4, sharing energy substrate throughout their network in accord with local requirements. Integration provides a distinct advantage over isolation: connected mitochondria support energy deficits, maintaining electrochemical gradients for ....

Protocol

Six-month-old (n=3) adult male DBA/2J [Wild-type (WT)] (RRID: IMSR_JAX:000671) and D2.B10-DMD^D2.mdx/J [D2.mdx (mdx)] (RRID: IMSR_JAX:013141), while three to five-month-old (n=8) adult male and female B6; D2-Tg(S100-EGFP)^1Wjt/J (S100B) (RRID: IMSR_JAX:005621) [also pooled with WT given no differences from DBA] were used from an ongoing colony of investigator-maintained mice. The overall experimental workflow for TA and GM injections, tissue processing, and imaging is summarized in Figure 1. An additional D2.mdx mouse was investigator-donated for tSC imaging in mdx (n=1). All animal procedure....

Results

Mitochondrial networks displayed expected reductions in connectivity
Representative 3D fluorescent images of TA muscle confocal scans in two groups, WT and mdx, are presented (Figure 2). In comparison with the WT group, mitochondrial connectivity appears reduced in mdx mice compared to the WT (Figure 2A–D). Greater numbers of small mitochondrial fragments were observed in mdx mice [<100 µm²] (WT, 17.3 ± 3.5%; m.......

Discussion

The location of mitochondria within cellular structures, as well as their morphology, correlates with mitochondrial health and metabolic demand²¹. Given the clinical relevance of mitochondrial health, coupled with divergent respiration kinetics and morphologies of different cell types, an easily applied, confocal microscopy-based method for the evaluation of mitochondrial morphologies in tSCs was developed after first evaluating the method in skeletal muscle, providing information on mitochondrial.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-in-1 Whole Animal System	Aurora Scientific, Ontario, Canada	1300A	Muscle funciton system with analysis software
AIVIA	Leica Microsystems, Bellevue, WA, USA	15	AI driven image analysis software
anti-laminin	MilliporeSigma	L9393-.2mL	myofiber boarder label
B6; D2-Tg(S100-EGFP)1Wjt/J Mouse Strain	Jackson Labs, Bar Harbor, ME	JAX:005621	S100β
Cryostar NX50 Cryostat	Epredia, Kalamazoo, MI, USA	https://www.epredia.com/products/histology-instruments/cryotomy/cryostar-nx50	Cryostat
D2.B10-DMDD2.mdx/J Mouse Strain	Jackson Labs, Bar Harbor, ME	JAX:13141	D2.mdx
DBA/2J Mouse Strain	Jackson Labs, Bar Harbor, ME	JAX:00671	WT
Dimethyl Sulfoxide (DMSO)	Fisher Scientific; Hampton, NJ, USA	BP231-100	Mitochondrial stain reconstitution reagent
Hoechst 33342	Thermo Fisher Scientific	H1399	Cell-permeant nucleic acid stain
ImageJ/Fiji	National Institutes of Health, Bethesda, MD, USA	SCR_002285	2D analysis software
Invitrogen ProLong Gold antifade reagent with DAPI	Fisher Scientific, Hampton, NJ, USA	P36941	Mounting medium
KimWipe	Kimberly-Clark, Irving, TX, USA	06-666A	Laboratory wipes
Leica LAS_X software	Leica Biosystems, Wetzlar, Germany	SCR_013673	Confocal software
MitoView Fix 640	Biotium	70075-50ug	Far-red fluorescent mitochondrial stain
NIS-Elements Advanced Research software	Nikon, Melville, NY, USA	SCR_014329	Cross sectional area analysis software
Permount Mounting Medium	Fisher Scientific, Waltham, MA 02454, USA	#SP15-100	General mounting media
Prism 9 software	GraphPad Software, La Jolla, CA, USA	RRID: SCR_002798	Statistical analysis software
Rhodamine (TRITC)	Fisher Scientific; Hampton, NJ, USA	AP132RMI	Secondary antibody
Stellaris 5 White Light Confocal Microscope and corresponding deconvolution software	Leica Microsystems, Wetzlar, Germany	158301313:158201310:158004752: 158301312:158301314:158401100: 158209011:158301130:158204510: 158301120:158401140:158204511: 15500332:15506428:15506517:155 13859:15521522:15525226:155252 32:15525314:15555009:15555017: 15555046:27100022:155933660: 155933666:155933668:158000640 :158002407:158003150:158004141 :158004201:158004421:158004423 :158200681:158202140:158203114: 158203200:158203220:158204201: 158204510:158204709:158301200: 9I_LL_STELLARIS_C:9T-CLSM-APP _CLASSC:9WE_LAS_405:9WE_ LAS_WLL:9WE_LL_STELLARIS_C	Confocal microscope
SZ61	Olympus, Breinigsville, PA, USA	sz61	Stereo microscope
Tissue-Plus O.C.T. Compound	Scigen, Fischer Scientific, Hampton, NJ, USA	23-730-571	Optimal cutting temperature compound
Visikol I	Sigma-Aldrich, St. Louis, MO, USA	H1-30	Tissue clearing solution 1
Visikol II	Sigma-Aldrich, St. Louis, MO, USA	H2-30	Tissue clearing solution 2
α-bungarotoxin 552	Biotrend, Koln, Germany	00014	Acetylcholine receptor dye