5 ethynyl 2 deoxyuridine edu

BrdU/EdU double-labeling was carried out according to Houlihan et al. (37 (link)). Briefly, pregnant females were injected intraperitoneally with BrdU, (Millipore-Sigma) (50 mg/kg body weight) and 1.5 hours later with the same dose of 5-Ethynyl-2’- deoxyuridine (EdU, Millipore-Sigma) and sacrificed after 0.5 hours. For EdU single-labeling, pregnant mice were injected intraperitoneally with EdU (50 mg/kg body weight) at E14.5 and sacrificed after 0.5 hours, 24 hours, or 4 days. The obtained mouse brain slices were stained for Edu immunohistochemistry using the Click-iT Edu kit (C10338,Thermo Fisher Scientific).

The freshly sorted cells were seeded in non-adherent 24-well plates (1 × 10⁵) and incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU; MilliporeSigma, Copenhagen, Denmark) for 24 h. Subsequently, the cells were fixed with 3.7% formaldehyde for 15 min and permeabilized in 0.5% Triton X-100 in PBS for 20 min. The cells were stained using the Click-iT EdU 488 Proliferation Kit (MilliporeSigma, Copenhagen, Denmark) following the manufacturer’s protocol. Next, cells were washed twice in 3% BSA/PBS and blocked for 1 h in 2% BSA/2% FBS in PBS. Cells were incubated in the dark for 20 min with 150 µL CD44-PE (1:5000) (Miltenyi Biotec) and counterstained with 10 µg/mL DAPI (MilliporeSigma, Copenhagen, Denmark) for 10 min. Cells (200–600) were analyzed using a Leica DM 2000 LED microscope (Leica Microsystems, Copenhagen, Denmark). Quantification was performed manually using the ‘cell counter plugin’ in ImageJ version 1.50i (National Institute of Health, Bethesda, MD, USA).