The RNA probe was transcribed in the presence of [35S]-uridine 5′-[α-thio]triphosphate (specific activity 1000–1500 Ci/mmol;
New England Nuclear, Boston, MA, USA). In situ hybridization was carried selleckchem out as described (Hurd, 2003) by applying the labelled probe to the brain sections at a concentration of 2 × 103 cpm/mm2 of the coverslip area overnight at 55°C in a humidified chamber. Two adjacent sections from each subject were studied. The slides were then apposed to Imaging Plates (FUJIFILM Corporation, Tokyo, Japan) along with 14C-standards (American Radiolabelled Chemicals, St Louis, MO, USA). Films were developed with a phosphoimaging analyzer (FLA-7000), and images were analyzed using the MultiGauge software (FUJIFILM Corporation). We have adopted the nomenclature of Paxinos & Franklin (2001) to describe the organization of the developing mouse brain. In addition, we have relied on the nomenclature introduced by Bons et al. (1998) for the adult mouse lemur to identify brain areas in the developing grey mouse lemur brain. find more A comprehensive list of abbreviations of neuroanatomical structures can be found in supporting Table S1. Recent findings demonstrate that scgn is
a CBP identifying neurochemically heterogeneous subsets of neurons in adult rodent, primate (Mulder et al., 2009b) and human forebrain (Attems et al., 2007). However, it remains unknown whether scgn is expressed during neurodevelopment. We assessed scgn mRNA levels in the mouse cerebral cortex (including hippocampus; Fig. 1A) and amygdaloid complex (Fig. 1A1) by qPCR analysis (supporting Fig. S1) during mid- and late-gestation, and in neonates. We established that pallial scgn mRNA expression was robust by E14.5, Resveratrol whilst moderate to low between E16 and P2 in the developing mouse neocortex and hippocampus (Fig. 1A). In contrast, scgn mRNA levels in the amygdaloid complex remained largely stable until birth with a marked decline being apparent after P1 (Fig. 1A1). Within the framework of the Human Protein Atlas program (Uhlen et al., 2005; Mulder et al., 2009a), we have generated antibodies to
> 8000 proteins, including a polyclonal antibody recognizing a phylogenetically conserved scgn epitope (Mulder et al., 2009b). Here, we confirmed that this antibody recognized a single protein target in samples prepared from neonatal mouse forebrain that is identical in size to that seen in adult brain (Fig. 1B; supporting Fig. S2), and corresponds to scgn’s calculated molecular weight of 32 kDa (http://www.ensembl.org). We explored whether scgn is expressed in the developing central nervous system at the protein level by detecting scgn protein upon loading fetal and neonatal forebrain lysates (40 μg/lane) on denaturing SDS-PAGE (Fig. 1C). The developmental dynamics of scgn mRNA expression suggest that this CBP may be transiently expressed by select cell populations in the fetal brain. Alternatively, scgn+ cells may be born by ∼E14.