Mice at embryonic day 12.5 (E12.5) and postnatal day 0 (P0) were fixed with
paraformaldehyde (PFA) in PBS at 4°C overnight. Next, whole embryos and brains at P0 were
cryoprotected in 30% sucrose and embedded in Tissue-Tek OCT compound (Sakura Finetek,
Tokyo, Japan). Frozen sections were prepared at a thickness of 20 µm
using a cryostat. For immunohistochemistry, sections were blocked with 10% normal goat
serum plus 0.3% Triton X-100 in PBS for 1 h at room temperature. The sections were
incubated with anti-MafB antibody (1:500, IHC-00351, Bethyl Laboratories, Montgomery, TX,
USA) overnight at 4°C. After washing, slides were incubated with Alexa Fluor
594-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA) for 1 h at room
temperature. Fluorescent images were acquired using a Biorevo BZ-9000 fluorescence
microscope (Keyence, Osaka, Japan). For assessment of efficiency of Cre-mediated
recombination, 4 sections were prepared from each sample, and tdsRed-positive areas were
measured with ImageJ software (NIH, Bethesda, MD, USA).
paraformaldehyde (PFA) in PBS at 4°C overnight. Next, whole embryos and brains at P0 were
cryoprotected in 30% sucrose and embedded in Tissue-Tek OCT compound (Sakura Finetek,
Tokyo, Japan). Frozen sections were prepared at a thickness of 20 µm
using a cryostat. For immunohistochemistry, sections were blocked with 10% normal goat
serum plus 0.3% Triton X-100 in PBS for 1 h at room temperature. The sections were
incubated with anti-MafB antibody (1:500, IHC-00351, Bethyl Laboratories, Montgomery, TX,
USA) overnight at 4°C. After washing, slides were incubated with Alexa Fluor
594-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA) for 1 h at room
temperature. Fluorescent images were acquired using a Biorevo BZ-9000 fluorescence
microscope (Keyence, Osaka, Japan). For assessment of efficiency of Cre-mediated
recombination, 4 sections were prepared from each sample, and tdsRed-positive areas were
measured with ImageJ software (NIH, Bethesda, MD, USA).