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.
Digoxigenin labeled ribonucleotides
Manufactured by Roche
Digoxigenin labeled ribonucleotides are a type of nucleotide that have been chemically modified to include a digoxigenin molecule. This modification allows the nucleotides to be used as labels for the detection and visualization of RNA in various molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti h3k4me3, by Abcam (2 mentions)
Nbt and bcip, by Roche (2 mentions)
Peroxidase conjugated anti digoxigenin and tyramide signal amplification tsa, by Thermo Fisher Scientific (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)
Alkaline phosphatase conjugated anti digoxigenin antibody, by Roche (1 mentions)
3 protocols using digoxigenin labeled ribonucleotides
1
In situ Hybridization and Immunostaining Protocol
2
In Situ Detection of IL-6 mRNA
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IL-6 RNA probe was established by reverse transcription of cDNA (for mouse IL-6: IMAGE ID 8861788, Source BioScience, Nottingham, United Kingdom; for human IL-6: IMAGE ID 3884652, Thermo Scientific. Waltham, MA) using digoxigenin-labeled ribonucleotides (Roche, Indianapolis, IN). Human tissue was obtained from banked pathology specimens under approval from Washington University Institutional Review Board. Sections of paraffin-embedded human tissue were rehydrated in xylenes followed by a series of alcohols. Fixed frozen mouse tissue prepared as above was fixed to glass slides with cold 4% paraformaldehyde in PBS. Tissue was then digested with 10 ng/ml proteinase K followed by treatment with acetic anhydride. The probe was allowed to hybridize overnight at 60°C in chambers moisturized with 50% formamide. Following hybridization, RNA was digested with 1 µg/ml RnaseA. RNA labeling was then detected using an alkaline phosphatase-conjugated anti- digoxigenin antibody (Roche) diluted 1∶2000. Intrinsic alkaline phosphatases were inactivated by incubation with 5 mM levamisole. Staining was detected with either Alkaline Phosphatase Kit I (Vector Laboratories, Burlingame, CA) or 75 µg/ml NBT combined with 175 µg/ml BCIP (Roche).
3
In situ Hybridization and Immunostaining Protocol
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+ Expand
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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