MANF expression in the hippocampus.

<p><b>A</b>: Rat pups were sacrificed at the indicated postnatal days. MANF expression in the hippocampus was determined by IHC. CA1 and CA3, Cornu Ammonis 1 and 3 region of hippocampus; DG, dentate gyrus; SUB, subiculum. <b>B</b>: Image of higher magnification showed CA1, CA3 and DG of hippocampus. Scale bar = 100 µm.</p

MANF expression in the substantial nigra.

<p><b>A</b>: Rat pups were sacrificed at the indicated postnatal days. MANF IHC in substantial nigra was shown. SNC, Substantia Nigra pars Compacta; SNR, Substantia nigra pars reticulata. <b>B</b>: Double-labeling immunofluorescent staining was performed to determine the localization of MANF (green) in the SNC of PD15 pups. Dopaminergic neurons were indicated by tyrosine hydroxylase (TH) (red). The indicated square in the top panel is shown at a higher magnification in the bottom panel. TH- positive and negative cells were indicated by open arrows and solid arrows respectively. Scale bar = 100 µm.</p

Detection of MANF.

<p><b>A</b>: Different amount of human recombinant MANF (1–20 µg) were loaded on a SDS-polyacrylamide gel and subjected to immunoblotting analysis using a rabbit anti-MANF antibody. <b>B</b>: Rat pups of PD15 were sacrificed and perfused. The brain was dissected, sectioned and subjected to MANF immunohistochemistry (IHC) using a rabbit anti-MANF antibody (1∶6,000) as described in the Material and Methods. Rabbit serum (1∶6,000) was used as a control. Images were taken from the cerebral cortex. Scale bar = 100 µm.</p

MANF expression in the olfactory bulb.

<p><b>A</b>: Rat pups were sacrificed at the indicated postnatal days. MANF expression in the olfactory bulb was determined by IHC. 1, Glomerular layer (GLL); 2, External plexiform layer (EPL); 3, Mitral cell layer (MCL); 4, Internal plexiform layer (IPL); 5, Granule cell layer (GL). Scale bar = 100 µm. <b>B</b>: Images of higher magnification showed the MCL. Scale bar = 25 µm. <b>C</b>: Double-labeling immunofluorescent staining was performed to determine the identity of cells expressing MANF. The images showed the expression of MANF (green), NeuN (red), GFAP (red) and DAPI (blue) in the MCL of PD15 rat pups. Scale bar = 25 µm.</p

MANF expression in the hypothalamus.

<p>Rat pups were sacrificed at the indicated postnatal days. MANF expression in the hypothalamus was determined by IHC. IHC images were taken from the indicated area shown above. <b>A</b>: MANF expression in supraoptic nucleus (SON). <b>B</b>: MANF expression in tuberomammillary nucleus (TMN). OPT, optic tract. Scale bar = 100 µm.</p

MANF expression in the cerebellum.

<p><b>A</b>: MANF expression in the cerebellum of PD6 pups was determined by IHC. DN, deep cerebellar nuclei; LC, locus coeruleus. Scale bar = 100 µm. <b>B</b>: Rat pups were sacrificed at the indicated postnatal days. MANF expression in the cerebellum was determined by IHC. Scale bar = 100 µm. <b>C</b>: Images of higher magnification showing MANF expression in the external germinal layer (EGL), molecular layer (ML), Purkinje cells (PCs) and internal granule layer (IGL). Scale bar = 100 µm. <b>D</b>: Double-labeling immunofluorescent staining was performed to determine the identity of cells expressing MANF. The images showed the expression of MANF (green), NeuN (red), GFAP (red), Calbindin (red) and DAPI (blue) in the cerebellum of PD15 rat pups. Scale bar = 25 µm <b>E</b>: Co-localization of MANF (green) with tyrosine hydroxylase (TH) (red) in the LC of PD15 rat pups. Scale bar = 100 µm.</p

Study of developmental expression of MANF in the cerebral cortex by immunoblotting analysis.

<p><b>A</b>: Rat pups were sacrificed at the indicated postnatal days (PD1-30). The cerebral cortex was dissected and homogenized. Equal amount of sample was pooled (3–5 pups per group) and subjected to immunoblotting analysis using an anti-MANF antibody described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090433#pone-0090433-g001" target="_blank">Fig. 1</a>. <b>B</b>: The relative expression of MANF was quantified by densitometric analysis. & p<0.05, && P<0.01 compared with PD1; *p<0.05, **P<0.01, ***P<0.001 compared with PD3, and ### P<0.001, compared with other groups.</p