Abstract
Evaluating the clinical potential of stem cell therapies for neurodegenerative diseases such as Alzheimer's requires careful longitudinal assessment and quantification of cellular survival, engraftment and integration. In preclinical animal models, fluorescence immunohistochemistry (fIHC) is the most common technique for analysis of transplanted cells and effects on host tissue. However, there are a number of significant limitations inherent to traditional fIHC, such as poor sensitivity to rare biomarkers, cost of antibody reagents and low signal-to-noise ratio, particularly in the context of significant auto-fluorescence in aged or diseased brains. Moreover, simultaneous detection of multiple biomarkers is restricted by species-specific antigen binding. In order to overcome these limitations, here we introduce the first application of the Perkin Elmer Opal fIHC technique to paraffin-embedded mammalian adult brain sections. Tyramide signal amplification means that fluorophore intensity is several fold greater than in traditional fIHC, while spectral unmixing corrects for auto-fluorescence without compromising biomarker signal. Furthermore, the Opal technique permits simultaneous quantification of up to eight co-localized biomarkers and is compatible with primary antibodies raised in the same species. This technique will greatly improve the study of stem cell biology, differentiation and integration through spatially selective protein phenotyping, and improve our understanding of interaction between host and donor cells in the pathological brain.
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