Click ittm edu imaging kit

Embryos from desired stages were incubated with 100 µM EdU/DMSO in NM for 30 min or 2 h at RT and then fixed immediately as described above. Samples were then used for FISH. After the final washes in PBTx, EdU incorporation was visualized using the Click-iT^TM EdU imaging kit with Alexa Fluor^TM 647 (Thermo Fisher Scientific, C10424) following the manufacturer’s protocol. Samples were mounted and imaged the same way as for FISH. Images and figures were made the same way as well. The counting of NvPrdm14d⁺ and EdU⁺ cells was performed on serial 1 µm sections through the image stack of whole embryos.

Lateral skin explants were taken from E13.5 wild-type embryos and placed on Nucleopore Track-Etch membranes (Whatman) floating on DMEM (Gibco) containing 10% FBS and 1× antibiotic/antimycotic solution (Gibco) at 37 °C and 5% CO₂. The explants were treated with either 10 µg/ml Mitomycin C or DMSO (vehicle) in the medium for 3 h, washed with 1× PBS, and cultured further in the medium above. Either directly at E13.5, after 1 day (E14.5) or after 2 days (E15.5), 20 µM EdU was added to the media for 2 h, then the explants were washed with 1× PBS and fixed in 4% PFA for 2 h at RT. After several washes with 1× PBS, the explants were removed from the membrane, cryoprotected in 30% sucrose overnight at 4 °C, and embedded in OCT for immunofluorescence staining (same protocol as for whole embryo sections except using 0.1% Triton^TM X-100 in PBS). For EdU analyses in embryonic mouse skin, the pregnant females at the corresponding time points were injected intra-peritoneally with EdU at 50 mg/kg and then anesthetized and sacrificed 3 h later for embryo collection, fixation, and OCT embedding as described above. The skin sections were treated according to the Click-iT^TM EdU imaging kit (Thermo Fisher) instructions before performing regular immunofluorescence staining.