Newly formed skeletal muscle fibers are prone to false positive immunostaining by rabbit antibodies.
Reports on muscle biology and regeneration often implicate immuno(cyto/histo)chemical protein characterization using rabbit polyclonal antibodies. In this study we demonstrate that newly formed myofibers are especially prone to false positive staining by rabbit antibodies and this unwanted staining is only recognized (1) by a negative muscle tissue control that does not harbor the protein to be examined (fx. from knockout mouse) or (2) by use of a nonsense rabbit antibody that has been prepared in the same way as the antibody of interest. However, many muscle immuno(cyto/histo)chemical studies only rely on controls that reveal non-specific binding by the secondary antibody and neglect that the primary rabbit antibody itself may cause false positive staining of the muscle. We suggest that reliable immuno-based protein detection in newly formed muscle fibers at least requires a nonsense rabbit antibody and optimally a negative muscle/cell control.
Andersen
Ditte C
DC
Department of Immunology and Microbiology, University of Southern Denmark, Odense C, Denmark.
Kliem
Anette
A
Schrøder
Henrik Daa
HD
Jensen
Charlotte H
CH
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D002454:Cell Differentiation; D002460:Cell Line; D005189:False Positive Reactions; D007150:Immunohistochemistry / Q000379:methods* / Q000592:standards*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000166:cytology / Q000276:immunology*; D009124:Muscle Proteins / Q000276:immunology; D032446:Myoblasts / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D011817:Rabbits
D000906:Antibodies; D009124:Muscle Proteins