Anti nicd cleaved notch1 val1744

Mice were euthanized following standard IACUC protocols. Specimens at E13.5, E15.5, E17.5, post-natal day 1 (P1), and 6-week old were collected and fixed overnight in 4% paraformaldehyde at 4°C for 24-48 h, demineralized in 0.5M EDTA for 3-14 days if required, dehydrated, embedded in paraffin wax, and serially sectioned at 7 μm. Histological sections were stained with haematoxylin and eosin (H&E). For in situ hybridization analyses, sections were hybridized to DIG-labeled RNA probes for detection of RNA transcripts. Sections were treated with 10 μg/mL of proteinase K and acetylated prior to hybridization with probe. DIG-labeled RNA probes were synthesized from plasmids containing full-length cDNA or fragments of Isl1, Shh, Fst, Etv5, Fgf9, Fgf10, Spry2, and Bmp4. Immunohistochemistry was performed according to standard protocols. Antigen retrieval was performed by boiling the slides in Trilogy (Cell Marque) for 15 min and cooled at room temperature for 20 min after removing paraffin and rehydration. Primary antibodies used were as follows: anti-ISL1 (1:200; ab20670; Abcam), anti-AMEL (amelogenin; 1:200; Abcam), anti-AMBN (ameloblastin; 1:200; Abcam), anti-CLDN1 (Claudin1; 1:200; Abcam), anti-NICD (Cleaved Notch1 (Val1744); 1:200; Cell Signaling Technology). Goat anti-rabbit or mouse AlexaFluor 555 secondary antibodies were used (1:500, Invitrogen).