Mice were sacrificed on day 21, at which time tumors and organs were collected

and blocked 5% non-fat dry milk in TBST. Blots were incubated with the antibody overnight at 4uC. Unbound antibody was removed by repeated washing with TBST. The blots were then treated with peroxidase-conjugated anti-rabbit antibody at a 1:10,000 dilution for 4 h at room temperature, respectively. The blots were developed with the ECL detection system according to the manufacturer’s instructions. Electron Microscopy Following immunoperoxidase visualization, sections were transferred to 0.1 M PB, postfixed for 30 min with 1% osmium tetroxide in 0.1 M PB, and washed several times in PB. Finally, sections were GSK126 dehydrated in a graded series of ethanol for 10 min each including block staining with 2% uranyl acetate in 70% ethanol, and flat embedded in Araldite CY212. Thin silver sections were contrasted with uranyl acetate and lead citrate and analyzed with a LEO 912 electron microscope equipped with a slow scan digital camera. Immunocytochemistry Immunoperoxidas. Immunocytochemical labeling was performed according to standard diamino benzidine/nickel immunoperoxidase protocols. Free-floating sections were treated with 1% sodium-borohydride in PBS for 15 min, washed with PBS and incubated in a solution containing 10% NGS, 0.3% Triton X-100, and 0.05% phenylhydrazine in PBS for 30 min. Primary antibody was diluted in 10% NGS, 0.3% Triton X-100 supplemented with 0.1% sodium azide and 0.01% thimerosal and incubated for 36 h at 8uC. After washing for 1 h in PBS and another hour in PBS containing 0.2% bovine serum albumin, the sections were incubated with biotinylated secondary goat anti-rabbit antibody for 24 h at 8uC. The sections were again washed as described above and further incubated for 6 h with an avidin-biotinyl-peroxidase-complex in PBS-BSA. After the final washing bound peroxidase was visualized in a solution containing 1.4 mM DAB, 10 mM imidazole, 6.6 mM nickel ammonium sulfate, and 0.15% H2O2 in 50 mM Tris/HCl buffer, pH 7.4. All sections were developed for 15 min. Labeled sections were mounted, dehydrated and cover-slipped with Entellan. Immunocytochemical controls. For negative controls, either primary or secondary antibodies were omitted. No staining was detected under these conditions. To further verify the specificity of anti-ADC and anti-Arg-labeling, purified antibodies were preincubated with the corresponding antigen at concentrations previously determined in a competitive ELISA assay was performed as described in detail previously. Briefly, freshly perfused brain tissue was sectioned at a thickness of 50 mm using a vibratome. Sections were rinsed in PBS, then incubated twice for 10 min in 20% sucrose diluted in 0.1 20363853 M phosphate buffer. 25939886 Subsequently, the sections were transferred on a plastic support and freeze-thawed using liquid nitrogen. After washing in PBS, sections were incubated in 0.1% sodium borohydride in PBS for 15 minutes. After washing in PBS, sections were pre-incubated for 30 minutes in a solution of 10% normal goat serum, 0.05% Triton-X-100 for permeabilization and 0.05% phenylhydrazine in PBS. Sections were then incubated for at least 36 hours at 4uC in the primary antibody solution, containing 10% NGS in PBS, 0.05% triton, 0.1% sodium azide and 0.01% thiomersal. Sections were washed in PBS, followed by pre-incubation with PBS-Albumin for 1 hour and incubation over night at 4uC with the biotinylated secondary antibody. After washing and pre-incubation in PBS-A for 1 hour, sections were incubated over night a