Fig 1.
Scheme of our staining method and the conventional immunostaining method.
The scheme in the panel on the left illustrates the conventional staining method. While this technique is widely used, it requires the production of several serial tissue sections to verify the distribution of each myosin heavy chain (MyHC) isoform. Hence, this method is time-consuming and laborious. Furthermore, multiple immunostaining of one section is difficult as all conventional anti-MyHC antibodies are mouse monoclonal antibodies. Meanwhile, the most time-consuming and laborious step in this process is to collate the serial sections, as it is challenging to locate the exact same regions within multiple large muscle cross-sections under a microscope. In contrast, our method is a very simple method. The staining is applied to individual tissue sections, and no secondary antibodies are required. It is therefore a highly efficient method for skeletal muscle fiber typing.
Table 1.
Synthesized peptides as an antigen for MyHC1, 2A, 2X or 2B.
Fig 2.
Evaluation of the validity of the myosin heavy chain (MyHC) monoclonal antibodies.
(A) We compared the efficacy of our newly developed antibodies with commercially available antibodies for immunofluorescence staining analyses of serial sections of skeletal muscle tissues. The images on the left show the staining patterns of each MyHC isoform in mouse sections, while the images on the right show the staining patterns in rat cross-sections. The distributions of MyHC1, MyHC2A, MyHC2X, and MyHC2B, which were stained with our newly developed antibodies, were nearly identical to those observed after staining with commercially available antibodies. Yellow arrowheads indicate representative examples of MyHC-positive fibers. The bars indicate 250 μm. The characters described at the upper-left of each image denote clone names. (B) Western blotting analysis using our newly developed monoclonal anti-MyHC antibodies. Mixtures of rat soleus and extensor digitorum longus muscle homogenates were separated by 8% SDS polyacrylamide gel electrophoresis [26], and blotted on a polyvinyl fluoride (PVDF) membrane. The specific immunoreactivities were confirmed by merging colloidal gold-stained total MyHC (pseudo colored magenta) and the bands detected by each antibody (pseudo colored green). Overlapping bands appear white in the merged images.
Fig 3.
Mouse and rat cross-sections stained using the staining method.
For these experiments, a single skeletal muscle section from a mouse (A) or a rat (B) was used. Cross-sections were obtained from calf muscles, including the gastrocnemius, plantaris, and soleus muscles, and immunopositive MyHC isoforms were visually classified as MyHC1 (white), MyHC2A (blue), MyHC2X (green), and MyHC2B (red). Images were taken with a 10× objective lens and assembled seamlessly using a tiling program, and high-resolution images covering whole muscle cross-sections were obtained. The panels on the right contain images that were magnified to determine the differences in distribution of the MyHC isoforms by examining the prevalence of each color. Bars indicate 1 mm in the whole muscle image, and 250 μm in the magnified images.
Fig 4.
Practical applications of the staining method.
One-step staining can provide additional data supporting the results of the electrophoretic detection of MyHC isoforms. (A) The dietary fat experiment: The electrophoretic image and the graph, published by Mizunoya et al. [23], contained in the panels at the left demonstrate that fish oil intake for four weeks induced significantly lower expression levels of the fast-type MyHC2B and higher expression levels of the intermediate-type MyHC2X protein in the extensor digitorum longus (EDL) muscles of rats than did the intake of soybean oil. Our one-step staining method succeeded in verifying the decrease in MyHC2B fibers and the increase in MyHC2X fibers in the plantaris muscles of fish oil-fed rats compared with soybean oil-fed rats. (B) The cold exposure experiment: The electrophoretic image and the graph, published by Mizunoya et al. [24], in the panels at the left show that cold exposure increased the prevalence of slow-type MyHC1 fibers in the soleus muscles of rats. Consistent with these findings, our staining method clearly depicts the predominance of MyHC1 in cross-sections of soleus muscles from these same cold-exposed rats. Bars indicate 500 μm in the whole images (upper) and 100 μm in the magnified images (bottom), respectively. Different superscripts indicate a significant difference between two groups (p < 0.05, one-way ANOVA; post hoc: Tukey–Kramer multiple-comparison test). *P < 0.05, compared to control group by unpaired Student’s t-test.
Table 2.
Percent distribution of pure MyHC1, 2A, 2X, 2B-positive and hybrid fibers in the rat plantaris muscles, based on immunofluorescent analyses shown in Fig 4A (right).
Table 3.
Percent distribution of pure MyHC1, 2A, 2X, 2B-positive and hybrid fibers in the rat soleus muscles, based on based on immunofluorescent analyses shown in Fig 4B (right).
Fig 5.
Practical applications of the staining method for cell cultures (isolated muscle fibers).
Muscle fibers isolated from the soleus, plantaris, or extensor digitorum longus (EDL) muscles of mice or rats were stained using the staining method. All bars indicate 100 μm.
Fig 6.
Practical applications of the staining method for analysis of longitudinal sections.
A longitudinal section from a rat plantaris muscle was stained using the staining method. The whole image provides a global view of the MyHC isoforms present in the section. The magnified images in panels 1 and 2 show the presence of hybrid fibers comprised of MyHC2X (green) and MyHC2B (red), or of MyHC2A (blue) and MyHC2X (green), as indicated by yellow and red arrows, respectively. Bars indicate 1 mm in the whole image and 250 μm in the magnified images, respectively.