• Coat a 3-cm petri dish with a thin layer (1.5-2mL) agarose and add BrdU labeling solution

• Dechorionate embryos in E3

• Transfer dechorionated embryos into BrdU solution (be careful to transfer as little liquid as possible)

    Note – Can also inject 5nL of a 10mM solution of BrdU into the yolk

• Swirl embryos to ensure exposure to labeling solution

• Incubate desired time (5 mins to overnight)

    Optional: Rinse and incubate in fresh E3 for desired time (chase)

• Fix embryos in BT, 3-4 hours at RT or overnight at 4ºC

• Incubate embryos in PBS + 1% triton X-100 for 3 hours RT or overnight at 4ºC

• Incubate in 4M HCl for 20 mins

• Wash 4 × 15 mins in PBS + 0.5% triton X-100

• Block for 1 hour at RT in immunofluoresence block

• Incubate with roche ά-BrdU antibody (1:100) overnight at 4ºC or 4 hours at RT

• Wash 4 × 15 mins in immunofluoresence block

• Incubate for 2 hours at RT in 1:500 alexafluor GάM secondary (488 or 546)

• Wash 4 × 15 mins in PBS + 0.5% triton X-100

• Visualize by fluoresence

BrdU labelling soltuion

E3 with 10mM BrdU in 10% DMSO in 3-cm dish 4.5mL E3 250μL 200mM BrdU stock 250μL DMSO

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