Fluorescent dyes and non-fluorescent ligands (haptens) are excellent affinity tags that can be recognized by labeled primary antibodies. They provide an alternative to the traditional signal amplification, as well as, expand the number of targets detected in multiplex experiments. Essentially all the methods that use biotin and avidin reagents are also possible using dyes/haptens, if corresponding anti-dye antibody is also available. Applications where anti-dye antibodies have been successfully used include DNA hybridization, immunohistochemistry, flow cytometry, ELISA, and western blotting. Their high specificity also makes them ideal reagents in applications where the signal detection is more difficult due to increased background, for example, non-specific detection of endogenous biotin in cells and tissue. Invitrogen anti-dye antibodies are designed to reliably detect fluorescent dyes and non-fluorescent ligands in various applications.
Invitrogen anti-dye antibodies are designed to dependably detect fluorophores and haptens in various applications.
In situ hybridization by tyramide signal amplification. a-Satellite probes to chromosomes 1, 15 and 17 were labeled by nick translation with biotin-11-dUTP, ChromaTide Texas Red-12-dUTP (Cat. No. C-7631) and ChromaTide Oregon Green 488-5-dUTP, respectively. Following simultaneous hybridization of all three probes, the biotinylated chromosome 1 probe was detected with HRP-streptavidin conjugate and Alexa Fluor 546 tyramide (TSA Kit #23). HRP activity from this first TSA detection step was then quenched by treatment with 1% hydrogen peroxide for 30 minutes. Lastly, the Oregon Green 488 dye-labeled chromosome 17 probe was detected with anti-fluorescein/Oregon Green antibody followed by HRP-conjugated goat anti-mouse IgG antibody and Alexa Fluor 594 tyramide (TSA Kit #5). HRP activity from this second TSA detection step was then quenched by treatment with 1% hydrogen peroxide for 30 minutes. The Texas Red dye-labeled chromosome 15 probe was then detected with Texas Red Polyclonal Antibody (Cat. No. A-6399) followed by HRP-conjugated goat anti-rabbit IgG antibody and Alexa Fluor 488 tyramide (TSA Kit #12). After counterstaining with Hoechst 33258, Pentahydrate (bis-Benzimide) (Cat. No. H1398, H3569, H21491), the images were acquired using filters appropriate for DAPI, FITC, TRITC, and Texas Red.
Multiplex FISH detection. The expression of Krupple (magenta) and rhomboid (orange) were detected using FISH Tag RNA Multicolor Kit, Alexa Fluor dye combination (Cat. No. F32956) combined in a whole mount Drosophila embryo. The expression of dpp was performed with a fluorescein-labeled RNA probe (ChromaTide fluorescein-12-UTP) and detected with TSA Kit #2 with Fluorescein/Oregon Green Polyclonal Antibody, HRP (Cat. No. A-21253).
Immunofluorescence using anti-nitrotyrosine antibody. Nitrated tyrosine residues in bovine pulmonary artery endothelial cells detected with rabbit anti-nitrotyrosine antibody. Fixed and permeabilized bovine pulmonary artery endothelial cells were treated with either degraded peroxynitrite (top panel) or 100 µM peroxynitrite (bottom panel) for five minutes at room temperature to induce protein nitration. Nitrated tyrosine residues were detected with our Nitrotyrosine Polyclonal Antibody (Cat. No. A-21285) and visualized with the green-fluorescent Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Cat. No. A-11008). Nuclei were counterstained with blue-fluorescent DAPI (Cat. No. D1306, D3571, D21490).