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Tools to analyze nuclear function,
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Fluorescence

ATTO (STED) Secondary Antibody Conjugates

anti-rabbit & anti-mouse secondaries for STED microscopy

For STED (STimulated Emission Depletion) microscopy using the Leica TCS STED microscope, the use of fluorescent ATTO 647N and ATTO 655 dyes are strongly recommended by Leica Microsystems. The fluorescent properties of these dyes meet the specifications required to perform STED microscopy with the 640 nm laser of the TCS STED system. Active Motif's Fluorescent Secondary Antibody Conjugates have been prepared using an optimized protocol that ensures the highest fluorescent intensity and stability. In addition, the ATTO dye conjugates have been maximally cross-adsorbed against IgG's of a variety of species to eliminate background caused by non-specific binding. These unique features make the Active Motif ATTO dye secondary conjugates ideal tools for STED microscopy, which will help in your explorations of sub-cellular space.

Confocal and STED microscopy images of cells stained with Histone H3 trimethyl Lys27 rabbit polyclonal antibody and ATTO 655 STED Goat anti-rabbit IgG
Figure 1: Active Motif ATTO 655 antibody conjugates in confocal and STED microscopy.

Histone H3 was stained with Histone H3 trimethyl Lys27 rabbit polyclonal antibody (Catalog No. 39155) and the ATTO 655 STED Goat anti-rabbit IgG (Catalog No. 15049) secondary antibody. The image on the left was prepared using a confocal microscope, while that on the right shows the histone proteins when visualized by STED microscopy. Images courtesy of Leica Microsystems, Germany.

The Leica TCS STED microscope can be upgraded with a second, 531 nm excitation laser that enables the use of one additional dye. Due to its long Stokes shift and other fluorescent properties, Leica recommends that Active Motif's Chromeo™ 494 and ATTO 647N dyes be used together for dual-color STED. This allows co-localization experiments below the resolution of the diffraction limit, helping in studies of the complicated nanostructure of the cell. In addition, dual-color STED will prove to be a useful tool for confirming protein-protein interactions that have been discovered by two-hybrid systems or fluorescence complementation assays in fluorescence microscopy. Dual-color STED enables one to demonstrate the interaction of both partners in their natural environment, instead of in modified or artificial, in vitro systems.

Comparison of nuclear structures visualized by dual color microscopy images with Chromeo 494 (green) and ATTO 647N fluorescent dyes using confocal and STED microscopes
Figure 2: Pre- and post-synaptic marker proteins visualized by STED microscopy.

The localization of pre- and post-synaptic marker proteins visualized in the dendrites of nerve cells. The pre-synaptic protein Bassoon was stained by ATTO 647N (red), while the post-synaptic protein Homer was stained with Chromeo 494 (green). The dendrite structure has been visualized by GFP and is presented as a blue overlay of a classical confocal picture. This STED image is courtesy of Dr. W. Zuschratter, IfN Magdeburg, Germany.

For additional information on ATTO STED antibody conjugates, please visit the Fluorescent Secondary Antibody Conjugates page.

To download the STED Microscopy Products Profile, please click here.

Active Motif's primary alpha Tubulin and H3K4me3 antibodies and its Chromeo 494 and ATTO 647N fluorescent secondary antibodies in STED microscopy
Figure 3: Active Motif's primary antibodies and fluorescent secondary antibodies in STED microscopy.

HeLa cells were stained with alpha Tubulin mouse monoclonal antibody (Clone 5-B-1-2) and Chromeo 494 Goat anti-mouse IgG (Catalog No. 15032). Histone H3 was stained with Histone H3 trimethyl Lys4 rabbit polyclonal antibody (Catalog No. 39159) and the ATTO 647N (STED/GSD) Goat anti-rabbit IgG (Catalog No. 15048) secondary antibody. The STED image is courtesy of Leica Microsystems, Germany.