Spatial Distribution of Shank3 mRNA in Murine Brain

In situ hybridization was performed according to general methodology, as described previously (Julien et al., 2008 (link), 2009 (link); Delay et al., 2014 (link)). DNA oligonucleotides were labeled with ³³P-dATP (PerkinElmer) using a three-terminal deoxynucleotidyl transferase enzyme kit (New England Biolabs). The reaction was conducted at 37°C for 2 h, and labeled oligonucleotides were purified with the QIAquick Nucleotide Removal Kit (QIAGEN). We used three different oligonucleotide mixes to hybridize to specific regions in the murine Shank3 mRNA (NM_021423.4). The first mix corresponds to sequences located before the ankyrin domain (nucleotides 337-296, 413-372, and 483-440); the second mix corresponds to sequences within the ankyrin domain (nucleotides 653-609, 1106-1061, and 1254-1209, which are deleted in the Shank3^Δex4-9 allele); the third mix corresponds to sequences after the ankyrin domain, within the proline-rich domain (nucleotides 3484-3440, 3693-3649, and 4127-4081). Prehybridation and hybridization conditions were performed as described by Julien et al. (2009) (link). Hybridized, 12 µm brain sections were exposed to Kodak Biomax MR films for 20 d. Nonspecific hybridization was considered negligible, as determined by adding a 100-fold excess of unlabeled probes.