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. 2010 Sep 10;14(1):98–115. doi: 10.3109/10253890.2010.512376

Figure 3.

Figure 3

Representative ISHH photomicrographs of gene expression in brain and pituitary of male Avpr1b KO and wild-type mice. Riboprobes corresponding to the following sequences were used: 5-HT1a receptor (Htr1a) (bp1230–1597 of Genbank Accession number NM_008308), 5-HT2a receptor (Htr2a) (bp1884–2492 of Acc#NM_172812), 5-HT2c receptor (Htr2c) (bp1435–1965 of Acc#NM_008312), brain-derived neurotrophic factor (Bdnf) (bp310–822 of Acc#AK017559), cannabinoid CB1R (Cnr1) (bp728–1040 of Acc#Y18374), Crh (bp101–686 of Acc#205769), type 1 Crh receptor (Crhr1) (bp160–528 of Acc#NM_007762), glucocorticoid receptor (Nr3c1) (bp439–958 of Acc#X04435), Oxt (bp1753–1865 of Acc# M88355), Avp (bp2966–3388 of Acc# M88354), intronic Avp (bp1965–2406 of Acc#M88354) and pro-opiomelanocortin (Pomc) (bp145-620 of Acc#V01529). The intronic Avp probe was targeted towards intron 1 of the Avp/neurophysin II gene to reflect heteronuclear RNA expression levels. These probes were obtained by PCR using genomic DNA, or brain (Crhr1, Crh, Bdnf, Avp and Oxt) or pituitary (Pomc) cDNA from 129 Sv mouse tissue as template. Restriction sites were incorporated into the 5′ ends of PCRprimers to facilitate cloning of the PCRproduct into vector pGEM4Z. The integrity of all riboprobes was verified by DNA sequencing. ISHH on 12 μm sections with 35S-labelled riboprobes was performed as in ref. (Lolait et al. 2007a). Sections were exposed to X-ray film (Hyperfilm MP, Amersham; GE Healthcare, Little Chalfont, UK) for hours (Pomc, Avp & Oxt) to weeks. Sections hybridised with the corresponding sense probes gave negligible, background staining (not shown). Brain regions shown include the nucleus accumbens (NA), PVN, supraoptic nucleus (SON) and the HIP. Sections of brain hybridised with the Nr3c1 probe were cut at a greater pitch around a mediolateral axis to obtain both HIP and PVN in one slice. Photomicrographs were taken at 6.3 × magnification and resized for publication (except Oxt, Avp and intronic Avp probes which are magnified appropriately to show the PVN or PVN/SON in greater detail).