2A). Immunoblot analyses using the obtained antibody detected a major band of 68 kD and
a smear from 68 to 95 kD in cDNA-transfected COS-7 cells, but not in mock-transfected and non-transfected COS-7 cells (Fig. 2B). The 68 kD band agrees well with the predicted molecular mass of rat Gpnmb, and the smear is most likely a glycosylated form. Furthermore, the antibodies recognized two main bands of 68 and 150 kD in a crude membrane fraction prepared from the entire brain (Fig. Inhibitors,research,lifescience,medical 2C). These bands were completely abolished by preadsorption of the antibody with the peptide used for immunization (Fig. 2C). Figure 2 Antibody validation. (A) COS-7 cells that were transfected with an expression plasmid for Gpnmb (Gpnmb) or an empty vector (Mock) and non-transected cells (-) were stained with anti-Gpnmb
antibody and visualized with FITC-conjugated Inhibitors,research,lifescience,medical secondary antibody … Gpnmb-IR in normal rat brain Cerebral cortex Gpnmb-IR was selleckbio observed in all selleck chemical layers of the cerebral cortex, but staining was most prominent in layers II and III (Fig. 3A). IR was abolished by preadsorption of the primary antibody with the peptide used for immunization (Fig. 3B). Some of the Gpnmb-IR cells appeared to have fine processes (Fig. 3C, D). Double immunofluorescence staining revealed that Gpnmb-IR cells in layers II and III were positive for Inhibitors,research,lifescience,medical the microglia/macrophage markers OX42 (Fig. 4A) and IB4 (Fig. 4B). Although less frequently, some Inhibitors,research,lifescience,medical Gpnmb-IR cells were co-stained for the radial glial lineage marker RC2 (Fig. 4C). In contrast, no co-staining was observed with antibodies
to the astrocyte lineage markers glial fibrillary acidic protein (GFAP; Fig. 4D) and protein S-100β (Fig. 4E) and the neuronal marker NeuN (Fig. 4F). This tendency was the same in other layers, except that occasional co-staining with NeuN was detected in layer VI (Fig. S1). Figure 3 Distribution of Gpnmb-IR in Inhibitors,research,lifescience,medical rat cerebral cortex. (A, B) Layers I–VI of the cortex. Sections obtained from adult rats were stained with anti-Gpnmb antibody before [ A, ads (-)] or after [ B, ads (+)] adsorption with the antigenic peptide and then … Figure 4 Characterization of Gpnmb-IR cells in cortical layers II–III with multiple markers. Sections were double-stained for Gpnmb (FITC, green) and the indicated markers (Texas Red, red). Note that Gpnmb-IR cells are co-stained with OX42, IB4, and RC2 … Hippocampus Gpnmb-IR was observed Anacetrapib throughout the hippocampus (Fig. 5A). IR was abolished by the primary antibody that was preadsorbed with the peptide used for immunization (Fig. 5B). IR in the CA1 segment (Fig. 5C) and dentate gyrus (Fig. 5F) was stronger than that observed in the CA2 and CA3 segments (Fig. 5D, E). With double fluorescence staining, Gpnmb-IR cells co-stained with OX42 or IB4 were observed in the polymorphic cell layer (Fig.