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-labeling experiments targeting small-caliber axonal segments. Nevertheless, the 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 simplify the conversion of FICC to EM by employing the unique properties of tyramide 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 segments was visualized by the application of a fluorescent secondary antibody, with the other tagged via a biotinylated antibody. Next, an ABC kit was used, followed by the simultaneous application of fluorophore- tyramide and biotin-tyramide. After temporary mounting for fluorescent digital photomicroscopy, sections were incubated in ABC and reacted with diaminobenzidine before EM analysis. Double-labeling fluorescent immunocytochemistry with TSA clearly delineated damaged axonal segments. In addition, these same axonal segments yielded high-quality EM images with discrete electron-dense reaction products, thereby providing a simple and reproducible means for following fluorescent analysis with EM.