Newly formed skeletal muscle fibers are prone to false positive immunostaining by rabbit antibodies

• Published in: Acta histochemica Vol. 113; no. 1; pp. 68 - 71
• Main Authors: Andersen, Ditte C., Kliem, Anette, Schrøder, Henrik Daa, Jensen, Charlotte H.
• Format: Journal Article
• Language: English
• Published: Germany Elsevier GmbH 2011
• Subjects: Animals; Antibodies - analysis; Antibodies - chemistry; Antibodies - immunology; Cell Differentiation; Cell Line; False Positive Reactions; False positive staining; Immunochemistry; Immunohistochemistry - methods; Immunohistochemistry - standards; Mice; Muscle Fibers, Skeletal - chemistry; Muscle Fibers, Skeletal - cytology; Muscle Fibers, Skeletal - immunology; Muscle Proteins - immunology; Myoblasts - chemistry; Myoblasts - cytology; Myoblasts - immunology; Rabbit polyclonals; Rabbits; Skeletal muscle regeneration; Skeletal muscle regeneration; Rabbit polyclonals; False positive staining; Immunochemistry
• ISSN: 0065-1281; 1618-0372
• DOI: 10.1016/j.acthis.2009.08.001

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.