Templates for the synthesis of riboprobes against mouse GAD67, CB1R, and DAGLα mRNA were generated by polymerase chain reaction (see Table). Nucleotide sequencing revealed 100% homology for the amplified template fragments to previously reported sequences. Sense and antisense riboprobes were generated by in vitro transcription in the presence of 35S-CTP using T7 or SP6 RNA polymerase, purified, and reduced to approximately 100 bp by alkaline hydrolysis to increase the effectiveness of tissue penetration (13). Standard hybridization procedures were performed as previously described (13). Following hybridization, sections from all mice for a given comparison were exposed to BioMaxMR film (Kodak, Rochester, NY) for 24 hours (GAD67), 48–72 hours (CB1R), or 36 hours (DAGLα). Tissue sections processed by in situ hybridization for CB1R mRNA were subsequently coated with NTB2 emulsion (Kodak) using a mechanical dipper (Auto-dip Emulsion Coater, Ted Pella, Redding, CA), exposed at 4°C, then developed using D-19 (Kodak), and counterstained with Cresyl violet. Specificity of the hybridization signal produced by each probe was confirmed by the findings that each antisense probe produced the expected distinctive laminar pattern of expression (4,14,15) and by the absence of labeling with sense probes.
