Nonisotopic colorimetric ISH was performed as described previously24 with some modifications such as a reduction in proteinase K concentration. Briefly, following cryosectioning of fresh-frozen samples at 20µm, tissue sections were fixed, acetylated, and subsequently dehydrated. Digoxigenin-based riboprobe labeling coupled with TSA amplification and alkaline-phosphatase-based colorimetric detection was used to label target mRNAs in expressing cells. Riboprobes were designed to overlap probe designs for homologous genes in mouse in the Allen Developing Mouse Brain Atlas (http://www.developingmouse.brain-map.org/). FISH was run as previously dd that high resolution images were captured on an Olympus Fluoview 1000 Confocal Microscope at 60× magnification.
