Vt1000s vibrosector

Mice were fixed by transcardiac perfusion with freshly-prepared 4% paraformaldehyde in PBS (Invitrogen). Brains were dissected, and 100 μm sections were prepared using a Leica VT1000S vibrosector. Slices were transferred into PBS with 0.5 μg/mL sodium azide (Sigma), then blocked with PBS supplemented with 3% horse serum (Invitrogen) and 0.3% Triton X-100 (Sigma) during 1 h, and incubated overnight at 4°C with the primary antibodies. After three washes with PBS/0.1% Triton X-100, slices were incubated in PBS for 1 h at room temperature and incubated 2 h at room temperature with the appropriated secondary antibodies. Sections were again washed three times with PBS/0.1% Triton X-100, stained with Hoechst (bisBenzimide H 33258, Sigma) or DAPI (Sigma) for 60 min and washed twice in PBS. The sections were next mounted on a Superfrost slide (Thermo Scientific) and dried using a brush before adding Glycergel mounting medium (Dako). Imaging was performed using either a Zeiss LSM780 or LSM880 confocal microscope controlled by the Zen Black software (Zeiss).

Embryos were fixed by transcardiac perfusion with freshly-prepared 4% paraformaldehyde (Sigma). Brains were dissected, and 100 mm sections were prepared using a Leica VT1000S vibrosector. Slices were transferred into PBS with 0.5 μg/mL sodium azide (Sigma), then blocked with PBS supplemented with 3% horse serum (Invitrogen) and 0.3% Triton X-100 (Sigma) during 1 h, and incubated overnight at 4°C with the following primary antibodies: chicken GFP (Abcam, 1:2,000), rabbit Pax6 (Covance, 1:1000), Sox2 (Santa Cruz Biotechnology, 1:500), PCNA (Millipore, 1:200), phospho-Histone H3 (Abcam, 1:100), Tbr2 (Abcam, 1:500), Neurod2 (Abcam, 1:500), or mouse β3-tubulin (Tuj1 epitope, Covance, 1:1000). After three washes with PBS/0.1% Triton X-100, slices were incubated in PBS for 1 h at room temperature and incubated 2 h at room temperature with the appropriated Alexa 488 (1:1,000, Molecular Probes), Cyanine 3 or Cyanine 5 (1:400, Jackson ImmunoResearch) secondary antibodies. Sections were again washed three times with PBS/0.1% Triton X-100, stained with Hoechst (bisBenzimide H 33258, Sigma) for 5 min and washed twice in PBS. The sections were next mounted on a Superfrost slide (Thermo Scientific) and dried using a brush before adding Glycergel mounting medium (Dako). Imaging was performed using a Zeiss LSM780 confocal microscope controlled by the Zen Black software (Zeiss).