Muscle Phenotyping Stains
Many different staining techniques are used in the analysis of muscle tissue. The preferred method for a project depends on the type of data that must be collected.
Hematoxylin and Eosin for Morphology
Hematoxylin and eosin staining. Pink: myofibers. Purple: nuclei.
Effects on Contralateral Muscles after Unilateral Electrical Muscle Stimulation and Exercise
(http://dx.doi.org/10.1371/journal.pone.0052230).
Routine H&E staining, while fast and inexpensive, lacks the specificity needed for automated myofiber detection. Manual editing of the boundaries between fibers is needed.
Immunofluorescence for Morphology
From: Multi-Parametric MRI at 14T for Muscular Dystrophy Mice Treated with AAV Vector-Mediated Gene Therapy (http://dx.doi.org/10.1371/journal.pone.0124914.g005).
In this image, red indicates laminin, green indicates dystrophin, blue indicates the dna in nuclei.
Ideally, identification of the borders of individual myocytes can be accomplished by immunostaining of dystrophin or laminin. This can be combined with nuclear staining. Since myofibers frequently appear bundled together, having a specific label to indicate the boundary of each greatly increases the automation analysis.
ATPase Staining for Fibertypeing
Images are serial top to bottom. From: Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse (https://doi.org/10.1371/journal.pone.0016908).
ATPase staining performed on serial tissue sections can be used to identify fiber types. Alkaline (pH 10.8) medium stains type II fibers darkly. Acid (pH 4.5) medium stains type I fibers darkly and IIa lightest. Acid (pH4.3) medium stains type I fibers darkly and type IIa and IIb lightest.
ATPase staining can be used with automated myofiber detection because of the strong contrast between stained and unstained myofibers. Some editing will still be required to separate adjacent stained fibers. Unstained fibers will not be analyzed unless they are manually drawn.
Immunohistochemical Staining for Fibertyping
Images are paired top to bottom. From: Unilateral Muscle Overuse Causes Bilateral Changes in Muscle Fiber Composition and Vascular Supply (https://doi.org/10.1371/journal.pone.0116455).
The upper row shows mAb A4951 antibody staining for type I fibers. The bottom row shows mAb N2.261 antibody staining for type IIa fibers.
NADH-TR Staining for Fibertyping
Images are serial top to bottom. From: Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse (https://doi.org/10.1371/journal.pone.0016908).
The purple coloring is concentrated in the mitochondria. Type I fibers stain more darkly than type II. The uneven color of the stain across the myofiber makes NADH staining problematic for automated myofiber detection.
SDH Staining for Oxidative Potential
From: The Relationship between Muscle Fiber Type-Specific PGC-1α Content and Mitochondrial Content Varies between Rodent Models and Humans (https://doi.org/10.1371/journal.pone.0103044)
Succinate dehydrogenase is an enzyme complex in mitochondira that’s part of the Krebs cycle. It’s indicative of the oxidative capacity of a myofiber. SDH staining itself is not widely used for fibertyping, rather it is analyzed in conjunction with other staining methods that do determine fibertype.
Multi-channel Fluorescent Staining for Fibertyping
From: Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis
(https://doi.org/10.1371/journal.pone.0035273).
Multi-channel fluorescent staining. Red: type IIB. Blue: type I. Green: type IIA. Purple: IIX / IIAX.
While more expensive and technically complex to perform, multi-channel immunofluorescence requires only a single section physical section and significantly simplifies imaging and automated fiber typing.