Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Summary

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

Abstract

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p > 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Introduction

It has been known for some time that skeletal muscle is composed of single fibers of many types¹. Initially, two groups of fibers were characterized based on their contractile properties and named, appropriately, slow-twitch (type I) and fast-twitch (type II). These categories were further distinguished on the basis of fiber metabolism. Since type I fibers are rich in mitochondria and reliant on oxidative metabolism, they were elucidated by robustly positive nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) diaphorase² or succinate dehydrogenase (SDH)³ staining. By contrast, type I....

Protocol

All procedures involving mice were approved by the University of Colorado-Anschutz Medical Campus Institutional Animal Care and Use Committee (91813(05)1D).

1. Day 1: Primary (1°) Immunostaining with Bovine Serum Albumin (BSA) Blocking

1. Air-dry frozen sections of mouse hindlimb muscle (e.g., tibialis anterior, soleus) mounted on charged slides for ~ 30 min¹¹. Draw a border around the sections using a hydrophobic barrier PAP pen.
2. Place ~ 250 µL of 5% BSA/phosphate buffered saline (BSA/PBS) on each slide to block non-specific antibody binding. Incubate slides at room temperature for 1 h.

Results

Hindlimb muscles (i.e., tibialis anterior, soleus) dissected from a male C57BL/6 mouse of unknown age were flash frozen by sinking a plastic mold containing the muscle in OCT compound in liquid nitrogen-cooled isopentane. Then, using a cryotome, 8-10 µm serial sections were cut at -20 °C and transferred to different positively-charged glass slides¹².

We chose tibialis anterior and soleus because .......

Discussion

Here, we have provided useful direction for the identification of skeletal muscle fiber types. In doing so, we describe a novel algorithm for analysis of the data.

Since our results largely confirm those of previous reports^5,8,10 and reflect our own manual measurements, the algorithm appears to be accurate. Still, we encountered some infrequent experimental pitfalls including unclear fiber borders res.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Serum Albumin	Sigma Aldrich	A9418-100G	5% in PBS
Hydrophobic Barrier Pap Pen	Scientific Device Laboratory	9804-02
Microscope Slides	Globe Scientific	1358W
Coverglass	Fisher Scientific	12-544-E
Immumount	Thermo Scientific	9990402
Nail Polish	L'Oreal
Nikon Eclipse TE-200 Inverted Fluorescence and Brightfield Microscope		Discontinued
SPOT RT/KE	SPOT Imaging Solutions	RT940
Dell Optiplex
BA-F8 Primary Antibody	Developmental Studies Hybridoma Bank at the University of Iowa		monoclonal mouse IgG2b; 1:50
SC-71 Primary Antibody	Developmental Studies Hybridoma Bank at the University of Iowa		monclonal mouse IgG1; 1:50
BF-F3 Primary Antibody	Developmental Studies Hybridoma Bank at the University of Iowa		monoclonal mouse IgGM; 1:50
6H1 Primary Antibody	Developmental Studies Hybridoma Bank at the University of Iowa		monoclonal mouse IgGM; 1:50
Alexa Fluor 594 anti-IgG2b	Invitrogen	A21145	goat anti-mouse; 1:200
Alexa Fluor 488 anti-IgG1	Invitrogen	A21121	goat anti-mouse;1:200
Alexa Fluor 594 anti-IgGM	Invitrogen	A21044	goat anti-mouse;1:200
OCT	Sakura Finetek	4583
isopentane	Fisher Scientific	O3551-4	cool with liguid nitrogen
PBS	Fisher Bioreagents	BP665-1	10x, dilute to 1x
Kim wipes	Kimberly-Clark	06-666A