Int J Physiol Pathophysiol Pharmacol 2010;2(1):29-35.
Original Article Improving the specificity of immunological detection in aged human brain tissue
Anyang Sun, Mei Liu, Guoying Bing
The Methodist Hospital Research Institute and Department of Radiology, Weill Cornell Medical College, Houston, TX 77030, USA; Institute of Neurobiology, Fudan University, Shanghai, 200032, PR China; Department of Anatomy and Neurobiology, University of Kentucky, Lexington, KY 40536-0098, USA
Received November 9, 2009; accepted November, 2009; available online December 2, 2009
Abstract: Immunological analyses of aged human brain tissues are widely used in characterizing the physiology or pathophysiology of brain aging or neurological diseases such as Alzheimer’s disease. The primary antibodies used in immunological detection mainly originate from rabbit and mouse species. In the present study, we showed an unexpected cross-immunoreactivity between anti-rabbit immunoglobulin G and diffuse lipofuscinassociated protein in aged human brain samples. In immunoblotting analysis of aged human brain samples, anti-rabbit secondary antibody alone produces a sharp band of approximately 180 kDa, whereas anti-mouse antibody does not show this cross-reaction. Immunohistochemical characterization of cross-immunoreactivity found that the crossreactive protein(s) were mainly associated with diffuse and weak autofluoresence in the cytoplasm of neuronsThis nonspecific cross-immunoreavctivity produces sufficient intensity of non-specific immunostaining signals that are easily mis-recognized as specific immunereactivity, then can generate misleading data. The above nonspecific crossreactivity with anti-rabbit secondary antibody in aged human brain tissue could be significantly reduced or abolished by pretreating tissues with sodium borohydride or adding 0.5% Tween- 20 into the secondary antibody buffer. When these modifications in the protocol, specific immunoreactivity (such as phospho-tau pT231) was unaffected, even slightly improved. Our study suggests that caution should be taken when performing immunological analyses on aged human brain samples with rabbit polyclonal antibody, and that modification of the experimental protocol is generally required to minimize the aforementioned nonspecificity. (IJPPP911001).
Key words: Immunoblotting, immunohistochemistry, aging, human brain tissues, nonspecificity