In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Summary

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Abstract

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

Introduction

Skeletal muscle regeneration is the result of the interaction between adult stem cells (Muscle Satellite Cells, MuSCs) and other cell types that are involved in the regenerative process. Muscle homeostasis and functionality are maintained by the combined signals arising from the muscle niche and systemic cues^1,2. Throughout the lifetime, changes in the MuSC functionality, the muscle niche, and the systemic cues have been reported, leading to the decline of functional capacities in the elderly³. MuSCs are set in a niche beneath the basal lamina and, upon muscle injury, are activated to r....

Protocol

Mice were bred and maintained according to the standard animal facility procedures, and all experimental protocols were approved by the Animal Welfare Assurance and the internal Animal Research Ethical Committee according to the Italian Ministry of Health and complied with the NIH Guide for the Care and Use of Laboratory Animals.

1. Muscle Injury and the In Vivo Block of Autophagic Flux

1. Muscle injury.
   1. To induce acute skeletal muscle injury, inject 20 µL of 10 µM cardiotoxin (CTX) stock directly in the left tibialis anterior (TA) muscle of 2 month-old C57BL/6J mice that weigh approx....

Results

This protocol describes an efficient in situ method to detect autophagy in MuSCs during muscle regeneration.

CTX In Vivo Treatments:

Use CTX to induce muscle damage in TA muscles and use unperturbed muscles as controls. Since autophagy is highly dynamic, block the autophagic flux by performing IP injections of CLQ (Figure 1).......

Discussion

This protocol describes how to monitor autophagy in skeletal muscle stem cells during compensatory muscle regeneration. Several antibodies for the co-staining of LC3 and MyoD were tried, and the ones that work in mouse tissue sections and create successful results are listed here (see Materials Table). The permeabilization with methanol (see step 3.2.2) is highly recommended for successful staining.

The limitation of this protocol is linked to the intrinsic variability of the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6J	The Jackson Laboratory	000664	WT mice
Cardiotoxin 1	Latoxan	L8102
Millex-VV	Merck Millipore	SLVV033RS	Syringe Filter Unit, 0.1 µm, PVDF, 33 mm, gamma sterilized
Chloroquine diphosphate salt	Sigma-Aldrich	C6628	Caution: Harmful if swallowed
BD Micro-Fine + 0.5 mL	BD	324825
Tissue-Tek O.C.T. compound	Sakura Finetek	25608-930
Tissue-Tek Cryomold Intermediate	Sakura Finetek	4566
2-Methylbutane	Sigma-Aldrich	277258
Hematoxylin Solution, Harris Modified	Sigma-Aldrich	HHS32
Eosin Y solution, alcoholic	Sigma-Aldrich	HT110132
o-Xylene	Sigma-Aldrich	X1040	Caution: Flammable liquid and vapour; May be fatal if swallowed and enters airways; Harmful in contact with skin; May cause respiratory irritation; Causes serious eye irritation
Paraformaldehyde (PFA)	Sigma-Aldrich	P6148	Caution: Flammable solid; Harmful if swallowed; Causes skin irritation; May cause an allergic skin reaction; Causes serious eye damage; May cause respiratory irritation; Suspected of causing cancer
DPBS, no calcium, no magnesium	Thermo Fisher Scientific	14190-094
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A7030
Glycerol	Sigma-Aldrich	G5516
Eukitt - Quick-hardening mounting medium	Sigma-Aldrich	3989
AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)	Jackson ImmunoResearch	115-007-003
LC3B Antibody	Cell signaling Technology	2775
Monoclonal mouse anti-MyoD (concentrated) clone 5.8A	DAKO - Agilent Pathology Solutions	M3512
Laminin-2 (α-2-chain) monoclonal antibody	Enzo Life Sciences	4H8-2
Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)	Life technologies	A11008
Alexa Fluor 594 Goat Anti-Mouse IgG (H+L)	Life technologies	A11005
Alexa Fluor Goat Anti-Rat IgM Antibody	Life technologies	A21248
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Thermo Fisher Scientific	D1306