3% Triton X-100, three changes, 5 min each; (6) ExtrAvidin-Peroxidase (1:1000; Sigma) in PBS containing 0.3% Triton X-100 for 30 min at RT; (7) PBS containing 0.3% Triton X-100, five changes, 5 min each; and (8) working learn more solution of the metal-enhanced diaminobenzidine (DAB) substrate kit (Thermo Scientific). After six rinses in distilled water, sections were mounted on untreated clean glass slides and covered with mounting medium Inhibitors,research,lifescience,medical (Aquatex; Merck, Darmstadt, Germany) and a glass cover slip. Photomicrographs were obtained using a light microscope (BZ-8000; Keyence, Osaka, Japan). Negative controls were obtained by preadsorbing antibodies with an excess (30 mM) of
the synthetic peptides. Multiple-label immunofluorescence Sections (16 μm) were prepared by the same method as for immunoperoxidase staining and sequentially incubated overnight at 4°C with rabbit anti-Gpnmb antibody (1 μg/mL) and mouse monoclonal antibodies in the blocking Inhibitors,research,lifescience,medical buffer; the details and final concentrations are given in Table 1. After rinsing, sections were incubated for 1 h at RT with a mixture of appropriate fluorescence-conjugated secondary antibodies (Table 1) in the blocking solution. Sections were examined
under a Inhibitors,research,lifescience,medical Keyence BZ-9000 microscope using OP-66836 BZ filter GFP-BP (excitation, 440–470 nm; emission, 535–550 nm), OP-66838 BZ filter TexasRed (excitation, 540–560 nm; emission, 630–660 nm), and OP-66834 BZ filter DAPI-BP (excitation, 340–360 nm; emission, 450–460 nm). Table 1 List of antibodies used in this study Staining with isolectin B4 (IB4) Sections were incubated with biotin-conjugated IB4 (1:100; Sigma) during primary antibody incubation and with Texas Red-conjugated streptavidin (1:100; GE Inhibitors,research,lifescience,medical Healthcare) during secondary antibody reaction. Results Gpnmb mRNA expression in rat brain To examine whether Gpnmb mRNA was expressed in
rat CNS, we first performed RT-PCR Inhibitors,research,lifescience,medical analysis. Primers were designed to distinguish between the amplified product from cDNA and an amplified product derived from contaminating genomic DNA. As shown in Fig. 1A, sense and antisense primers were made to recognize exons 6 and 11, respectively. PCR products from cDNA and genomic DNA were predicted to be 993 bp and 4.4 kb, respectively. Furthermore, specificity of PCR products was confirmed by Southern blot analysis using an internal probe (Fig. Linifanib (ABT-869) 1A). Gpnmb mRNA expression was detected in all brain regions examined; GAPDH cDNA was used to confirm the integrity of RNA preparations (Fig. 1B). Figure 1 Reverse transcription-polymerase chain reaction (RT-PCR) analysis of Gpnmb mRNA in CNS of adult rats. (A) Schematic representation of the recognition sites of Gpnmb-specific PCR primers (arrows), the predicted sizes of the amplification products, and … Antibody validation To examine Gpnmb expression at the protein level, we generated a polyclonal antibody against rat Gpnmb by immunizing rabbits with a synthetic peptide corresponding to the C-terminal region.