The biotin- and digoxigenin-labeled probes were detected consecutively using the dual-color tyramide signal amplification (TSA) procedure. The biotin-labeled probe was detected by streptavidin-HRP (1:100, Perkin Elmer). Then, the first amplification reaction was carried out under a coverslip by applying 50 μl of Cy3-tyramide (1:50, Perkin Elmer) for 20 min at room temperature. Thereafter, the slides were soaked in blocking reagent (Perkin Elmer) for 15 min at room temperature to block the remaining peroxidase activity. Subsequently, the digoxigenin-labeled probe was detected by anti-digoxigenin-HRP (1:200, Roche), followed by TSA amplification using FITC-tyramide (1:50, Perkin Elmer). Finally, the slides were washed in 4× SSC, dehydrated in an ascending ethanol series, and mounted in Vectashield (Vector Laboratories).
Anti digoxigenin hrp
Manufactured by Roche
Anti-digoxigenin-HRP is a reagent used in molecular biology and immunohistochemistry applications. It consists of an antibody against digoxigenin conjugated to the enzyme horseradish peroxidase (HRP). This conjugate can be used to detect and localize digoxigenin-labeled biomolecules, such as nucleic acids or proteins, through a colorimetric or chemiluminescent reaction catalyzed by the HRP enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 tyramide, by PerkinElmer (1 mentions)
Fitc tyramide, by PerkinElmer (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Blocking reagent, by PerkinElmer (1 mentions)
Streptavidin hrp, by PerkinElmer (1 mentions)
Tsa fluorescence system, by PerkinElmer (1 mentions)
Metamorph analysis, by Molecular Devices (1 mentions)
Apoptag plus in situ apoptosis fluorescein detection kit, by Merck Group (1 mentions)
A11120, by Thermo Fisher Scientific (1 mentions)
Ta180004, by OriGene (1 mentions)
Nel701a001kt, by PerkinElmer (1 mentions)
Megascript kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti digoxigenin hrp
1
Dual-Color Tyramide Signal Amplification
2
Fluorescent mRNA in situ Hybridization
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Fluorescent mRNA in situ hybridization was performed as described, with digoxigenin labeled probes [52 (link)]. Briefly, hybridized probe was detected with anti-digoxigenin-HRP (Roche), using fluorescein-labeled tyramide as a substrate (TSA Fluorescence System, Perkin Elmer). Embryos were mounted in 70% glycerol/PBS.
3
Quantifying Apoptosis in Diabetic Rat Retina
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Frozen rat retinal sections were assessed for DNA strand breaks by TUNEL assay as per the manufacturer's instructions (ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit, EMD Millipore, Billerica, MA). In some cases where FITC-BSA or GFP cells were injected, a modified HRP detection system with anti-digoxigenin HRP (1∶500; Roche, Indianapolis, IN) was adopted. In each experiment, adjacent sections incubated without TdT served as control. The total number of TUNEL-positive cells in diabetic rats was normalized to total nuclear cells using MetaMorph analysis (Molecular Devices, Sunnyvale, CA) and shown as a percentage of non-diabetic animals that received saline injection.
4
Multi-modal Developmental Gene Expression
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Digoxigenin-UTP-labeled antisense RNA probes were transcribed using MEGAscript Kit (Ambion) according to the manufacturer's instructions. Whole-mount in situ hybridizations were performed according to previously published methods (Ning et al., 2013 (link)). In particular, fluorescent in situ hybridization in immunostained embryos was conducted as previously described (Mao et al., 2021 (link)). Briefly, for the detection of cxcr4a and tie1 expression, Tg(nkx2.5:ZsYellow) embryos were first immunostained using affinity-purified anti-ZsYellow antibody (1:800; TA180004, Origene), and then subjected to in situ hybridization with digoxigenin-labeled cxcr4a or tie1 probe. Anti-digoxigenin-HRP (1:400; Roche, 11633716001) was used as primary antibody to detect the probes and the hybridization signals were visualized by incubating embryos with fluorescein tyramide (1:50; PerkinElmer, NEL701A001KT). For the detection of cxcl12b expression, Tg(sox17:GFP) embryos were fluorescently stained with anti-GFP (1:1000; A11120, Invitrogen) antibody. The resulting hybridization signals were similarly generated with cyanine 3 tyramide (1:50; PerkinElmer, NEL701A001KT).
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