Hoechst 22358

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).

For confocal microscopy, melanoma cells were seeded on sterile coverslips and infected with 2,000 pfu of pOKA, pOKA ORF62/71 S686/722A, and pOKA ORF62/71NLS mutants. Infected and uninfected control cells were fixed with 4% formaldehyde at 24 and 48 hpi. After blocking with 1% fish gelatin for 1 hr at RT, cells were incubated with a rabbit anti-ORF63 antibody and a murine monoclonal anti-ORF62 antibody. Cells were washed and incubated for 1 hr at RT with fluorescein isothiocyanate-labeled anti-rabbit and Texas Red-labeled anti-mouse secondary antibodies (Jackson ImmunoResearch, Inc.). Cell nuclei were counterstained with Hoechst 22358 (Invitrogen, Carlsbad, CA). Analysis was performed with a Leica TCS^SP2 confocal laser scanning microscopy (Heidelberg, Germany).