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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.
Simultaneous detection of expression of five genes in a whole mount Drosophila embryo by fluorescence in situ hybridization (FISH) with five RNA probes Go ›
Labeled paint probes hybridized to human metaphase chromosomes Go ›
Immunocytochemistry using Alpha-Tubulin Monoclonal Antibody, Mouse Go ›
Simultaneous detection of three gene targets in a whole-mount Drosophila embryo by fluorescence in situ hybridization. Go ›