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dehydrogenase, was imaged using a Pannoramic SCAN
™
(Caliper) slide
scanner fitted with a 20
objective. Next, the coverslips were removed
from the biopsies and the samples were labeled for biomarkers such as the
extracellular matrix protein laminin, MyHC-
, or TRMU (tRNA (5-
methylaminomethyl-2-thiouridylate)-methyltransferase), a mitochondrial
tRNA-modifying enzyme (
Uusimaa et al., 2011
), whose mutation leads
to reversible infantile respiratory chain deficiency (
Uusimaa et al., 2011
),
as well as neonatal hepatopathologies (
Fellman and Kotarsky, 2011;
Kemp et al., 2011
). In certain cases, the biopsies were colabeled for two bio-
markers (e.g., TRMU and MyHC-
b
b
) in addition to the colorimetric
enzymatic stain.
Excellent labeling of the biomarkers was achieved in the skeletal muscle
biopsies despite the previous processing of these samples for the colorimetric
enzymatic assays which had occurred several years (
>
5 years) previously
(
Fig. 7.12
). Notably, in this sample, there is correlation between NADH-
dehydrogenase labeling (blue, colorimetric) with MyHC-
b
, which is
expected, since Type I fibers, which express MyHC-
, are known to con-
tain a higher concentration of mitochondria than Type II fibers.
In another example, a human skeletal muscle biopsy clinically assayed for
NADH-dehydrogenase activity was fluorescently relabeled using antibodies
against MyHC-
b
and TRMU. In this case, CyteSeer
®
was able to segment
the image (identify fiber outlines) from the bright-field images of enzyme
activity and use those masks not only for the analysis of enzyme activity
but also for analyzing the fluorescent MyHC-
b
(red) and TRMU (green)
expression (
Fig. 7.13
). For this biopsy, the fibers that were positive for
MyHC-
b
showed a more intense colorimetric stain for NADH-
dehydrogenase activity and also for TRMU labeling. While the correlation
of NADH-dehydrogenase activity with MyHC-
b
is expected, based
upon the known preponderance of mitochondria in Type I fibers, the data
for TRMU, while preliminary in nature, may be the first suggesting a Type
I expression pattern for this protein.
In an additional example, a skeletal muscle biopsy from a patient with
mitochondrial disease was
b
labeled for SDH activity, TRMU, and
MyHC-
(
Fig. 7.14
). The biopsy featured several “ragged blue” fibers for
SDH labeling. Ragged blue fibers are equivalent to “ragged red” fibers visu-
alized with Gomori trichrome and the “ragged” phenotype is due to abnor-
mally high numbers of mitochondria in the subsarcolemmal space of fibers
that are typically deficient for COX activity (
Filosto et al., 2007; Reichmann
et al., 1996; Walker and Schon, 1998
). Interestingly, while ragged red fibers
b
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