Novel Application of Tyramide Signal Amplification (TSA): Ultrastructural Visualization of Double-labeled Immunofluorescent Axonal Profiles

Abstract

SUMMARY 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.

A novel closed-body model of spinal cord injury caused by high-pressure air blasts produces extensive axonal injury and motor impairments

“…Our findings for RMO14 and SBDP150 run counter to the observation that axons typically label for both markers in studies on rats examined 15 min to 3 h after TBI induced by moderate impact acceleration (Buki et al, 1999, 2000). It is likely that responses to different types and magnitudes of injury follow different time courses, with moderate impact acceleration TBI probably being more severe than our blast injury and its sequelae transpiring more quickly.…”

“…Although nearly 80% of SBDP+ axonal profiles were RMO14+ by 2 days, the early pathology we observed differs from that produced by moderate impact acceleration, where injured axons exhibit extensive SBDP150 and RMO14 double-labeling within 15 min (Buki et al, 1999, 2000). The latter findings have led to the inference that RMO14 labeling is a consequence of μ-calpain proteolysis of neurofilament sidearms (Buki and Povlishock, 2006).…”

Alarin stimulates food intake in male rats and LH secretion in castrated male rats

“…For immunofluorescent detection, slides were processed using the tyramide-amplification procedure (Stanarius et al, 1997, Stanarius et al, 1999, Toda et al, 1999, Wang et al, 1999, Buki et al, 2000, Bobrow and Moen, 2001). Briefly, the slides were incubated in affinity-purified rabbit anti-alarin (Santic et al, 2007) as primary antibody (1:100 in KPBS with 0.4% Triton-X) for 24 hr at room temperature.…”

Activation of the Hedgehog pathway in the mouse fetal ovary leads to ectopic appearance of fetal Leydig cells and female pseudohermaphroditism

“…Secondary antibodies used were FITC-, Rhodamine- or Cy3-conjugated donkey anti-Rabbit and FITC- or Rhodamine-conjugated donkey anti-Goat (all 1:200, Jackson Immuno Research, USA). When two primary antibodies from the same species were used, tyramide amplification combined with sequential immuofluorescence was performed following the technique described in Büki et al (Buki et al, 2000). Fluorescent images were captured using a Fast1394 QImaging Camera (QImaging, Canada) installed on a Leica Dmi 4000B microscope (Leica, Germany).…”