Evaluation of commercial dysferlin antibodies on canine, mouse and human skeletal muscle.
Immunostaining of muscle biopsy cryosections is a powerful tool for identifying protein deficiencies. For dysferlin, a protein associated with limb-girdle muscular dystrophy and Miyoshi myopathy, weak immunostaining of normal muscle has been a problem in reliably identifying dysferlin deficiency in human patients or dystrophic animals. Here we use skeletal muscle cryosections from dog, mouse and human to test several dysferlin antibodies under different conditions of fixation, and without fixation. NCL-Hamlet antibody (mouse monoclonal), following fixation in acetone/methanol, provided the strongest and most reliable staining in sections of human muscle as well as of dog and mouse muscle. Unlike animal tissue, unfixed human muscle also gave strong and reliable staining. NCL-Hamlet 2 gave good staining in all species. Epitomics (rabbit monoclonal) antibody gave good staining of all muscles, and did not stain muscle of dysferlin-deficient mice. However, it strongly stained muscle sarcolemma of patients with dysferlin deficiency, making the antibody less useful. Abcam antibody gave weak staining, and Santa Cruz antibodies did not immunostain muscle dysferlin in any species tested. NCL-Hamlet antibody was optimal for immunoblotting in all species. Use of select antibodies for immunostaining and immunoblotting, and optimization of immunostaining methods, should increase the sensitivity of detecting dysferlin deficiency in skeletal muscle.
Guo
Ling T
LT
Department of Pathology, University of California San Diego, La Jolla, CA, USA.
Moore
Steven A
SA
Forcales
Sonia
S
Engvall
Eva
E
Shelton
G Diane
GD
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D004285:Dogs; D000073939:Dysferlin; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*
D000906:Antibodies; C114839:DYSF protein, human; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins