In situ hybridization of a-satellite probes to human chromosomes 1, 15 and 17 detected 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 (C7631) 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, 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 Texas Red dyes.
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