In situ hybridizations and immunostaining were carried out according to standard protocols. The cDNA for pdm2 was PCR amplified using primers listed below and cloned into a pBSK+/− vector at EcoRI restriction site. Riboprobe was synthesized using T7 polymerase and digoxigenin labeled ribonucleotides (Roche). Alkaline phosphatase conjugated with anti-digoxigenin (Roche) and NBT and BCIP (Roche) were used to develop in situ hybridization. Peroxidase conjugated anti-digoxigenin and Tyramide signal amplification (TSA, Life Technologies) was used for fluorescent in situ hybridization (FISH). WIDs and EIDs were analyzed with a DMLB microscope and SPE confocal microscope (Leica). Primary antibodies used were: rabbit anti-H3K4me3 (1:1,000, Abcam/ab8580), mouse anti-En (1:25, DSHB/4D4) and mouse anti-CycA (1:100, DSHB/A12), mouse anti-BOSS (1:1,000)58 and rabbit anti-GFP (1:1,000, Santa Cruz Biotechnology/sc-8334). Fluorescently labeled secondary antibodies were from Life Technologies and Jackson Immunochemicals. Discs were mounted in SlowFade (Life Technologies) supplemented with 1 μM TO-PRO-3 (Life Technologies) to label nuclei. For all in situs and immunostainings around 10 imaginal discs were analyzed. All experiments were performed twice.
Peroxidase conjugated anti digoxigenin and tyramide signal amplification tsa
Manufactured by Thermo Fisher Scientific
Peroxidase conjugated anti-digoxigenin and Tyramide signal amplification (TSA) is a laboratory product that facilitates the detection and amplification of digoxigenin-labeled biomolecules. It combines a peroxidase-conjugated anti-digoxigenin antibody and a tyramide-based signal amplification system to enhance the sensitivity of target detection.
Automatically generated - may contain errors
Lab products found in correlation
Digoxigenin labeled ribonucleotides, by Roche (2 mentions)
Rabbit anti h3k4me3, by Abcam (2 mentions)
Nbt and bcip, by Roche (2 mentions)
Slowfade, by Thermo Fisher Scientific (2 mentions)
Spe confocal microscope, by Leica (2 mentions)
Dmlb microscope, by Leica (2 mentions)
Rabbit anti gfp, by Santa Cruz Biotechnology (2 mentions)
2 protocols using peroxidase conjugated anti digoxigenin and tyramide signal amplification tsa
1
In situ Hybridization and Immunostaining Protocol
2
In situ Hybridization and Immunostaining Protocol
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In situ hybridizations and immunostaining were carried out according to standard protocols. The cDNA for pdm2 was PCR amplified using primers listed below and cloned into a pBSK+/− vector at EcoRI restriction site. Riboprobe was synthesized using T7 polymerase and digoxigenin labeled ribonucleotides (Roche). Alkaline phosphatase conjugated with anti-digoxigenin (Roche) and NBT and BCIP (Roche) were used to develop in situ hybridization. Peroxidase conjugated anti-digoxigenin and Tyramide signal amplification (TSA, Life Technologies) was used for fluorescent in situ hybridization (FISH). WIDs and EIDs were analyzed with a DMLB microscope and SPE confocal microscope (Leica). Primary antibodies used were: rabbit anti-H3K4me3 (1:1,000, Abcam/ab8580), mouse anti-En (1:25, DSHB/4D4) and mouse anti-CycA (1:100, DSHB/A12), mouse anti-BOSS (1:1,000)58 and rabbit anti-GFP (1:1,000, Santa Cruz Biotechnology/sc-8334). Fluorescently labeled secondary antibodies were from Life Technologies and Jackson Immunochemicals. Discs were mounted in SlowFade (Life Technologies) supplemented with 1 μM TO-PRO-3 (Life Technologies) to label nuclei. For all in situs and immunostainings around 10 imaginal discs were analyzed. All experiments were performed twice.
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