Novel application of tyramide signal amplification (TSA): Ultrastructural visualization of double-labeled immunofluorescent axonal profiles

Fluorescent immunocytochemistry (FICC) allows multiple labeling approaches when enzyme-based techniques are difficult to combine, such as in double-la beling experiments targeting small-caliber axonal segments. Nevertheless, t he conversion of FICC to a product visible at the electron microscopic (EM) level requires labor-intensive procedures, thus justifying the development of more user-friendly conversion methods. This study was initiated to simp lify the conversion of FICC to EM by employing the unique properties of tyr amide signal amplification (TSA), which allowed the simultaneous targeting of a fluorescent tag and biotin label to the same antigenic site. Briefly, one of two antigenic sites typically co-localized in damaged axonal segment s was visualized by the application of a fluorescent secondary antibody, wi th the other tagged via a biotinylated antibody. Next, an ABC kit was used, followed by the simultaneous application of fluorophore-tyramide and bioti n-tyramide. After temporary mounting for fluorescent digital photomicroscop y, sections were incubated in ABC and reacted with diaminobenzidine before EM analysis. Double-labeling fluorescent immunocytochemistry with TSA clear ly delineated damaged axonal segments. In addition, these same axonal segme nts yielded high-quality EM images with discrete electron-dense reaction pr oducts, thereby providing a simple and reproducible means for following flu orescent analysis with EM.