A20012

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).