Digoxigenin dig 11 utp

Mouse Lhx8 fragment plasmid (nt 1022–1185 of the mouse cDNA) was a gift from Drs. M. Grigoriou and V. Pachnis [15 (link)]. Following linearization with NotI and HindIII respectively, antisense and sense control RNA probes were synthesized by in vitro transcription with T3 or T7 RNA polymerase (MAXIscript® T3/T7 In Vitro Transcription Kit, Ambion), per manufacturer instructions using Digoxigenin (DIG)-11-UTP (Roche, Germany). Whole mount in situ hybridization of E11.5 embryos was performed per prior methods[16 (link), 17 (link)]. Mandibular incisor and first molar tooth germs from CD-1 mice (Charles River Laboratories) at E12.5, E14.5, E16.5, E18.5, P1, P5, P7 and P21 were dissected in cold PBS (pH=7.4) and fixed with Z7 (0.5% w/v zinc chloride, 0.5% w/v zinc acetate, 0.05% w/v calcium acetate, 17.16 mM zinc trifluoroacetate, in 0.1M Tris-HCl, pH=7) for 30 min to 6 hrs at room temperature (RT) with gentle agitation. Tissues were transferred to 30% w/v sucrose and incubated at 4°C prior to OCT mounting. Frozen sections (10 μm) were cut for in situ hybridization[15 (link)]. NBT/BCIP from DIG Nucleic Acid Detection Kit (Roche) was used with constant color development for 24 hrs for all sections. Lhx8 transcripts were hybridized at 67°C with 1:5000 diluted anti-DIG-AP antibody (Roche). All animal experiments were approved by Columbia University IACUC.

Mouse Alx3 fragment plasmid (1,856-kb-cDNA cloned from E16 cDNA library), a generous gift from Dr. Frits Meijlink, was used to map Alx3 from E12.5 to P7. Following transformation and linearization with EcoRI or HindIII (New England BioLabs, Ipswich, MA, USA), antisense and sense RNA probes were synthesized by transcripting with Sp6 and T7 polymerases (MAXIscript® Sp6/T7 In Vitro Transcription Kit, Ambion, Austin, TX, USA), following manufacturer instructions using Digoxigenin (DIG)-11-UTP (Roche, Mannheim, Germany). The mandibular incisor and first molar tooth organs at E12.5, E14.5, E15.5, E16.5, E18.5, P1, P3, and P7 were harvested by gestation timing of CD-1 pregnant mice (Charles River, Newark, NJ, USA). The isolated tooth germs were dissected under surgical microscope in cold DEPC-treated PBS (pH=7.4), and processed for fixation with 4% paraformaldehyde for 24h at 4°C with gentle rotation. The isolated tooth germs were then exposed to 30% w/v sucrose and incubated at 4°C prior to mounting. Postnatal tooth organs were first decalcified with 0.5M EDTA (pH=8.0) and dehydrated with 30% w/v sucrose. Frozen samples at 8-pm thickness were sectioned for in situ hybridization. NBT/BCIP from DIG Nucleic Acid Detection Kit (Roche) was used to detect Alx3 expression. Alx3 transcripts were treated at 65°C with 1:5000 diluted anti-DIG-AP antibody (Roche).