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G55 cells were subcuta neously injected into SCID mice. Microbeads containing 1 × 106 WT or transfected PAE cells were implanted at the same site 7 days later. In the combination groups 1 × 106 PAE cells producing each inhibitor INNO-406 ic50 were injected. Each experimental group consisted of 5 animals. After 10 days, animals were sacrificed and tumors were ex cised and weighed. One half of each tumor was fixed in 10% formaldehyde and embedded in paraffin for immu nohistochemistry. The other half was frozen in liquid nitrogen and used for RNA isolation. Immunohistochemistry and immunofluorescence Paraffin embedded tissue samples were serially sectioned at a thickness of 5 um, and every 20th section was used for analysis. Tissue sections were consecutively stained with hematoxylin and eosin.<br><br> Blood vessels were visualized using murine polyclonal CD31 antibody. Monoclonal Ki67, polyclonal prolactin receptor and M30 CytoDEATH antibodies were purchased from LBH589 Dako, abcam and Roche Applied Science respectively. Immunhistochemical staining was performed as previously described. For double immunhistochemical analyses, M30 and PRLR anti bodies were visualized with Diaminobenzidine and 3 Amino 9 ethylcarbazole, respectively. A blocking step in between using the Avidin Biotin Block ing Kit was performed. For immunofluorescence detection of PRLR in G55 cells, 3× 105 cells were seeded on chamber slides and treated with CM or mixtures of CM from PAE WT, PAE Tum and PAE ES cells for 3 days. After fixation with cold ice methanol, staining was performed as pre viously described.<br><br> Microvessel density was quanti fied by counting CD31 オーダー LY2109761 positive vessels in 10 arbitrarily chosen visual fields per tumor from totally 4 to 5 tumors from each experimental animal group using AxioVision40 V4. 8 software. TUNEL assay of apoptotic cells For the in situ detection of fragmented DNA, tissue sec tions were subjected to terminal deoxynucleotidyl trans ferase dUTP nick end labeling using the in situ cell death detection kit, POD according to the manufacturer´s instructions. Nuclei were counterstained with haematoxylin. TUNEL negative nuclei were stained blue, while TUNEL positive nuclei were stained brown. RNA isolation and microarray analysis Frozen tumor samples were homogenized with a micro tissue disintegrator. Tissue homogenates were first treated with TriReagent for RNA Isolation succeeded by purification with the RNeasy Mini Kit following manufactures protocols.<br><br> Quality and concentration of isolated RNA was determined using the Agilent RNA 6000 Nano Kit and NanoDrop6000 Photometer. From each experimental animal group, three RNA samples were selected for further microarray analyses. Sense strand cDNA was generated from 100 ng total RNA using the Ambion WT Expression Kit. Procedures for labelling, fragmentation and hybridization were performed using the Terminal Labeling Kit and Hybridization, Wash and Stain Kit following Affymetrix protocols. All experiments were performed using Affymetrix Human Gene 1. 0 ST Array containing 28. 869 genes. Microarrays were scanned with the GeneChip Scanner 3000 7G using the GeneChip Command Console 3. 0.