Automated Methods for the Analysis of Skeletal Muscle Fiber Size and Metabolic Type - International Review of Cell and Molecular Biology - page 281

perinatal ( Richard et al., 2011 ); and an evaluation of the changes in fiber com-
position associated with MMP-9 knockout, utilizing antibodies against
MyHC Type I, MyHC IIB, and MyHC IIX ( Mehan et al., 2011 ).
The immunostaining method could be further advanced with the devel-
opment of isoform-specific antibodies made in species other than mice (rab-
bit, goat, and sheep) and with development of primary mouse monoclonal
antibodies directly conjugated to fluorophores representing distinct fluores-
cence channels. Both approaches will encourage the development of mul-
tiplexed assays in which multiple fiber types can be assayed on the same
skeletal muscle section.
4. SKELETAL MUSCLE METABOLISM
4.1. Characterization of muscle metabolic enzyme activity
via histochemical staining
It is possible to metabolically characterize skeletal muscle, on a fiber-by-fiber
basis, by performing assays to semiquantitatively evaluate the activity of met-
abolically relevant enzymes. To do this, fresh-frozen sections of skeletal
muscle are thawed and incubated with substrates that are specific for
enzymes such as COX, SDH, or NADH dehydrogenase, which are com-
ponents of Complex IV, Complex II, or Complex I, respectively, of the
electron transport system ( Fig. 7.2 ; Engel, 1970 ). In these assays, the final
reactant is an electron acceptor such as diaminobenzidine or nitro-blue
tetrazolium, which, when oxidized, precipitates as a colored product in a
fiber-specific manner (brown for diaminobenzidine or blue for nitro-blue
tetrazolium). In the context of determining muscle fiber types, SDH assayed
in this manner was widely adopted in the 1960s for visualizing muscle mito-
chondrial density and characterizing fiber types in a variety of contexts,
including rat diaphragm muscle ( Padykula and Gauthier, 1963 ), fetal and
newborn mice ( Wirsen and Larsson, 1964 ), and effects of denervation
( Bajusz, 1964; Cherian et al., 1966; Smith, 1965 ) or exercise ( Edgerton
et al., 1969; Schiaffino and Bormioli, 1973; Schiaffino et al., 1970 ).
With time, the conditions for histochemical staining of enzymatic activity
were optimized and many investigators contributed to this development. For
example, Old and Johnson (1989) optimized SDH and COX staining in sec-
tions of human skeletal muscle. Blanco et al. (1988) described histochemical
technique for quantitative determination of SDH activity in muscle sections;
this was reevaluated by Powers et al. (1993) who concluded that the method
was both valid and reliable in the determination of SDH activity in skeletal