| OCR Text |
Show 57 Drug treatments Embryos were treated at 18 hpf with 200mM NaClO3 (Sigma-Aldrich, USA) for 24 hours at 28.5°C. Treated embryos were fixed in 4% PFA overnight at 4°C. HS inhibition was confirmed by 10E4 and 3G10 antibody staining (USBiological, USA) and pea3 and lef1 in situs. SU5402 (Tocris, USA) treatment: inhibition of pea3 halo and dpERK in ext mutants and NaClO3-treated embryos (25μM – 42-48 hpf); ectopic protrusions (12.5μM - 24 hours); extl3/ext2 protrusions (40μM from 58-68 hpf); Wnt induction in ext mutants (28-34 hpf – 20μM). To induce Wnt with 2μM BIO (Tocris, USA) embryos were treated from 24 – 48 hpf. Bmp was inhibited by immersion in 10 μM Dorsomorphin (Tocris, USA) from 12 - 48 hpf. All solutions and controls contained 1% DMSO in 0.5X E2 medium. In situ hybridization and immunohistochemistry The full open reading frame of gpc1b, gpc4, sdc3 and sdc4 was amplified and inserted into the pCS2+ vector using standard molecular cloning techniques. Probes used: lef1, pea3, sef, axin2, fgf3, fgf10, fgfr1, dkk1b, atoh1a, cxcr4b and cxcr7b (Aman and Piotrowski, 2008), wnt10a (Lush and Piotrowski, 2014) and cxcl12a (Li et al., 2004). Cloning of s100t was performed using Fw 5’-ACCAGCAGTCATCTCACCTC-3’ and Rv 5’-CACACATTCAACAAAGACCATCG-3’ primers. patched1 probe was generated from a full length clone. Whole mount immunostaining was performed according to standard protocols. Primary antibodies used: 4D9-mouse (Engrailed) 1:6 (Hybridoma), 10E4-mouse 1:200 (USBiological), ZO-1-mouse 1:200. For Di-pErk1/2-mouse 1:100 (Sigma-Aldrich), Glyofix was used as fixative at 4°C. For 3G10-mouse 1:200 |
