In situ hybridization
Coronal sections were cut from the olfactory epithelia of an adult mouse (Figure 4) and a young (P6) C57BL/6 mouse. RNA in situ hybridization was carried out as described previously [15,53] with digoxigenin-labeled antisense riboprobes specific for the 3' UTRs of genes AY318555 (0.5 kb) and AY317365 (0.5 kb). Riboprobe sequences were generated by PCR using primer pairs 5'-TCTTCCAAACCTGGACCCCCC-3' and 5'-ATCTCTCCAGCACCTTACTTG-3' for AY318555 and primer pairs 5'-TAAGATGTAAGTGATAATTTAGATTACAGG-3' and 5'-TTTCTGCCTCAGCTATGACAG-3' for AY317365. Hybridization was carried out in 50% formamide at 65°C, and slides were washed at high stringency (65°C, 0.2 × SSC). The probes each hybridize to only one band on a Southern blot, indicating that each probe only detects one olfactory receptor gene. BLAST analyses show that the AY318555 probe is unique in Celera's mouse genome assembly (Release 13), and that the AY317365 probe is similar to only one other genomic region. This potential cross-hybridizing region is over 10 Mb from the nearest olfactory receptor coding region and is thus highly unlikely to be included in any olfactory receptor transcript. Low-power images are composed of three overlapping micrographs (40×) assembled in Adobe Photoshop 7.0.