Neuron, Volume 60 Supplemental Data Genomic Anatomy of the Hippocampus

Double fluorescent in situ hybridization (DFISH) is based on the Allen Institute’s colorimetric ISH protocol, which is described at http://brain-map.org/pdf/ABADataProductionProcesses.pdf and in Lein et al., 2007. Briefly, riboprobes are labeled with either digoxigenin (DIG) or dinitrophenyl-11-UTP (DNP; Perkin Elmer). The DNP-labeled probe and DIG-labeled probe are hybridized simultaneously, and the signal from each is amplified with tyramide sequentially using either anti-DIG-HRP with tyramide-biotin, or anti-DNP-HRP with tyramide-DNP. Signal is detected by labeling of biotin or DNP with streptavidin-Alexa-Fluor 488 (Invitrogen/Molecular Probes) or anti-DNP-Alexa-Fluor 555 (Invitrogen/Molecular Probes) respectively, and automated fluorescence microscopy.