Table 3.

Antibodies used routinely with consideration of CMD specific findings.

Antibody	Findings
Laminin α2	Absence (in muscle fibers and nerves, skin biopsy can also be used) = MDC1A Primary reduction = MDC1A, check laminin α5 (should be elevated) Secondary reduction = suggestive of dystroglycanopathy-check 2H6 (glycosylated αDG) labeling (in LGMD2I this reduction is seen on blots only) Partial reduction may need antibody against 300 kDa fragment to appreciate the reduction
Laminin β1 and γ1	Should be normal in all CMDs - serves as basement membrane control in laminin α2 deficiency
Laminin α5	There is secondary over-expression in MDC1A (note that regenerating fibers have higher expression, while moderate levels may be present in neonatal muscle biopsies)
α-Dystroglycan	Immunolabeling with antibody against glycosylated αDG (such as 2H6). Absence or virtual absence of immunolabeling = seen in severe CMD forms, including MEB, WWS, FCMD. Also seen in LG forms with Fukutin mutation. Incomplete or mild reduction = seen with abnormal αDG glycosylation of various severity, including LGMD presentations (unclear genotype/phenotype correlations, some will have FKRP mutations)
β-Dystroglycan	Should be normal in most CMDs - serves as a control for α-dystroglycan (some mild reduction may sometimes be seen) Marked reduction is exclusion criterion for CMD (seen in dystrophinopathies)
Collagen VI	Complete absence = suggestive of recessive UCMD Reduction from sarcolemma only, with good preservation of sarcolemma (Labeled with perlecan of collagen IV) = suggestive of dominant UCMD, some recessive missense mutations show similar behavior. Normal appearance does not exclude Bethlem in particular Cultured skin fibroblasts may be more sensitive for subtle changes.
Dystrophin	Absence-exclusion criterion for CMD = seen in DMD Some mild reduction - can be secondary in some dystroglycanopathies (no genotype correlation), if more prominent, suggestive of BMD Note that labeling with dys 2 may be non-specifically weak in some neonates (age related)
Sarcoglycans	Should be normal in all of the CMDs Reduction - exclusion criterion
Utrophin	Mild to moderate elevation of immunostaining may be non-specific, seen in regenerating fibers Consistent high levels of expression: exclusion criterion, suggestive of dystrophinopathy
Myosins	Co-expression of fast and slow isoforms in several fibers = suggests abnormal muscle, but is non-specific Predominance of slow fibers may occur in CMD and is nonspecific Presence of several fibers with developmental/neonatal myosin- may indicate regeneration, and/or delayed development, and/or non specific abnormality Note: in neonates a direct correlation of myosin immunolabeling with ATPase staining is difficult as the presence of neonatal myosin (in relation to immaturity) makes many fibers stain as type 2 but they may in fact react positively for slow myosin. The decline in the number of fibers with neonatal myosin in normal muscle is not known but by 3–6 months there are generally very few. The presence of many fibres with neonatal myosin at 1 yr is indicative of an abnormality