In situ hybridization of a-satellite probes to human chromosomes 1, 15 and 17 detec¬ted 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. C7631) and ChromaTide® Oregon Green® 488-5-dUTP (Cat. no. C7630), respectively. Following simul¬taneous hybrid¬ization of all three probes, the biotin¬ylated chromosome 1 probe was detected with HRP–streptavidin conjugate and Alexa Fluor® 546 tyramide (TSA Kit #23, Cat. no. T20933). 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 (Cat. no. A6421) followed by HRP-conjugated goat anti–mouse IgG antibody and Alexa Fluor® 594 tyramide (TSA Kit #5, Cat. no. T20915). 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 rabbit anti–Texas Red® antibody (Cat. no. A6399) followed by HRP-conjugated goat anti–rabbit IgG antibody and Alexa Fluor® 488 tyramide (TSA Kit #12, Cat. no. T20922). After counterstaining with Hoechst 33258 (Cat. no. H1398, H3569, H21491), the images were acquired using filters appropriate for DAPI, FITC, TRITC and the Texas Red® dye.