Prb 445p

Embryonic mice were decapitated and heads were fixed in 4% paraformaldehyde (PFA) in PBS at 4°C overnight. Postnatal animals were perfused and brains were dissected out, then fixed in 4% PFA in PBS at 4°C overnight. Parasagittal sections 7μm thick were prepared from fixed tissues embedded in paraffin. Hematoxylin and eosin (H&E), immunofluorescence, and immunohistochemical staining of tissue sections were performed as previously described (Park et al., 2016a (link); Park et al., 2016b (link)). Antibodies used were Yap (1:500; abcam ab56701), BLBP (1:200; Millipore ABN14), Calbindin (1:200; Sigma, C9848), S100β (1:200; Novus Biologicals, NB110–57478), GFAP (1:200; Thermo Scientific, RB-087), BrdU (1:500; abcam, ab6326), PCNA (1:250; Proteintech, 10205–2-P), pH3 (1:500; Millipore Sigma, 06–670), NeuN (1:250; Millipore, MAB377), Pax6 (1:200; Covance, RBP-278), Sox9 (1:200; Millipore, AB5535), Pals1 (1:200; Proteintech), Pan-Crb (1:200; (Cho et al., 2012 (link))), NMIIB (1:500; Covance, PRB-445P), N-Cadherin (1:500; BD, 610920), and β-Catenin (1:500; BD, 610153). Species-specific secondary antibodies conjugated to Alexa Fluor 488 (1:250; Invitrogen) or Cy3 (1:250; Jackson Immunochemical) were used for immunofluorescence. Nuclei were stained with Hoechst 22358 (1:500; Invitrogen).

Whole cerebellums from P0 Yap/Taz dCKO or Yap heterozygote/Taz CKO with Nestin-Cre and littermate control mice (Cre⁻) were lysed to obtain protein for Western blot analysis as previously described (Park et al., 2016b (link)). Briefly, proteins were separated on an SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. After blocking with 5% nonfat dry milk, the membrane was incubated with primary antibodies Yap (1:1000; Abcam ab56701), Taz (1:1000; V386 Cell signaling), Pals1 (1:1000; Proteintech, 17710–1-AP), β-Catenin (1:1000; BD, 610153), NMIIB (1:2000; Covance, PRB-445P), N-Cadherin (1:1000; BD, 610920) and GAPDH (1:4000; Proteintech 60004–1-Ig and 10494–1-AP). Protein signals were detected using chemiluminescence (ECL Kit, GE Healthcare).