Embryos were incubated in 0.5 mM (2′S)-2′-deoxy-2′-fluoro-5-ethynyluridine (EdU, Sigma T511293)) diluted in fish water. Embryos were anesthetized and fixed at 48 hpf in 4% paraformaldehyde with 1X PBS (phosphate-buffered saline) and 4% sucrose for 30 minutes at room temperature. After fixation, embryos were permeabilized in PBS + 0.5% TritonX100 for 30 minutes at room temperature, and brain tissue was dissected. To visualized EdU labeling, brains were incubated in a solution containing 10uM Cy5-azide (Lumiprobe A2020), 2 mM copper(II) sulfate (Sigma 45167), and 20 mM sodium ascorbate (Sigma A7631) for 1 hour at room temperature. Following incubation, brains were washed in PBS + 0.5% TritonX100 and processed for immunofluorescence using standard blocking and antibody incubations. The antibody used was chicken anti-GFP (1:250, Abcam) followed by goat anti-chicken 488 (Molecular Probes, 1:250).
T511293
Manufactured by Merck Group
The T511293 is a lab equipment product from Merck Group. It is a precision measurement device used for scientific applications. The core function of this product is to provide accurate and reliable measurements for laboratory experiments and research activities.
Automatically generated - may contain errors
2 protocols using t511293
1
EdU labeling of Zebrafish Brain
2
Visualizing DNA Replication in Animals
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Animals were fed EdU (Sigma #T511293) diluted in liver to 0.5 mg/ml and 1% DMSO. Following feeding, animals were incubated in 5g/L Instant Ocean until fixation. Animals were processed using the FISH protocol with a modified EdU labeling step after probe hybridization. Animals were incubated in the dark for 30 min in an azide click reaction containing 1% 100 mM CuSO4, 0.1% 10mM azide-fluorophore 545 (Sigma #MKCH3642), 20% 50 mM ascorbic acid from (+) sodium-L-ascorbate in PBS. Ascorbic acid was made fresh for each reaction. After EdU labeling animals were washed in PBSTx 3x and FISH protocol was continued.
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