5 ethynyl 2 deoxyuridine (edu)

Five-week-old mice were intraperitoneally injected with 50 mg/kg EdU (Tokyo Chemical Industry, Tokyo, Japan). Six hours later, the salivary glands were extracted, fixed with 4% paraformaldehyde, and embedded in paraffin. Paraffin sections (5 µm thick) were processed to detect EdU-positive cells, and cell proliferation was analysed using the Click-iT Plus Alexa Fluor Picolyl Azide Toolkit (Thermo Fisher Scientific). For analysis of cellular apoptosis, TUNEL staining was performed using the In situ Apoptosis Detection Kit (Takara Bio) according to the manufacturer’s instructions.

For detecting S-phase cells at various time points, 1 mg/ml EdU (Tokyo Chemical Industry, 100 µl) was injected into the abdominal cavity at 0 or 10 dpl, or 2 or 4 wpl. Newts were euthanized 1 h after EdU injection for ‘short-term labeling’, and perfusion fixed and postfixed with 4% paraformaldehyde in PBS. Cryosections were prepared as described above for immunohistochemistry. To detect M-phase cells, anti-pH3 antibody was used.

A thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU, Tokyo Chemical Industry) was intraperitoneally injected into a pregnant mouse (50 mg/kg body weight) at the indicated time points before or after TM injection. The brains were dissected from the mice at P0, and coronal sections of 16-μm thickness were prepared as explained above. The sections were antigen-retrieved as described above and immunostained with chicken anti-βGAL and donkey Alexa488-conjugated anti-chicken IgY antibodies. Subsequently, the incorporated EdU was detected in a solution of 0.1 M Tris (pH 7.6), 2 mM CuSO₄, 3 μM Alexa555 azide triethylammonium salt (A20012, Thermo Fisher Scientific), and 10 mM ascorbic acid for 40 min at room temperature. The numbers of neurons labeled for βGAL and those doubly labeled for βGal and EdU were manually counted for each mouse with a 40× objective lens (Plan-Apochromat) under a fluorescent microscope (Zeiss Axioplan2) with 38 HE (ex470/40, em525/50) and 15 (ex549/12, em590) filter sets. The exact numbers of animals and neurons used for quantification are shown in the figures and legends. The scattered plots (Figures 5C–5F and 7C–7F) were generated using the Prism8 software (GraphPad).