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 lipofuscin-associated protein(s) in aged human brain tissues. 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 localization of cross-immunoreactivity found that the cross-reactive protein(s) were mainly associated with diffuse and weak autofluorescence in the cytoplasm of neurons. This nonspecific cross-immunoreactivity produces sufficient intensity of non-specific immunostaining signals that are easily mis-recognized as specific immunoreactivity, and can generate misleading data. The above nonspecific cross-reactivity with anti-rabbit secondary antibody in aged human brain tissues could be significantly reduced or abolished by pretreating tissues with 1% sodium borohydride or/and adding 0.5% Tween-20 into the secondary antibody dilution buffer. When these modifications are included in the protocol, specific immunoreactivity (such as phospho-tau pT231) was unaffected, or 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
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