Methods Tissue samples Primary and selleck metastatic tumor tissues of different origin, among them 15 paired samples of human primary and metastatic lesions and various pre malignant lesions, were selected from the archives of the Depart ment of Pathology and Radiology of the Radboud Univer sity Nijmegen Medical Centre. Furthermore, non tumor related tissues were obtained. The study was per formed according to the guidelines of the Code for proper secondary use of human tissue in the Netherlands. Immunohistochemistry After deparaffinization and blocking of endogenous per oxidase activity, antigen retrieval was performed by treat ment with pronase according to standard protocols. Non specific binding sites were blocked by incubation with 20% normal horse serum.
Slides were incubated for 1 hr with single domain antibody A12, which was previ ously selected against a PLXND1 specific peptide. A12 was detected by sequential incubations with the mouse anti VSV G P5D4, biotinylated anti mouse IgG, and avidin biotin peroxidase complex. Peroxidase was visualized by the 3 amino 9 ethylcarbazole peroxidase reaction, with haematoxylin Inhibitors,Modulators,Libraries as counterstain. All incuba tions were performed at room temperature. Blood vessel origin was confirmed by endothelial stainings on serial sections with anti human CD31 antibody. In a selection of tissues, macrophage identity was con firmed by double staining for PLXND1 and CD68. In short, the above mentioned avidin biotin peroxidase pro cedure was used to detect PLXND1 via rabbit anti VSV G antiserum. Following visualization, avidin biotin was blocked according to standard protocols.
Inhibitors,Modulators,Libraries Slides were suc cessively incubated with normal horse serum, mouse anti human CD68 antibody overnight at 4 C, bioti nylated anti mouse IgG and avidin biotin Inhibitors,Modulators,Libraries alka line phosphatase complex at RT. AP was visualized with a mixture of naphthol phosphate, levamisole, and Fast Blue. PLXND1 mRNA In Situ Hybridization To explore whether the PLXND1 transcript is also specifi cally present in malignant tissues, we performed mRNA in situ hybridizations on 28 of the 158 paraffin embedded tissues analyzed by immunohistochemistry as pre viously described. In brief, digoxigenin labelled sense and antisense human PLXND1 RNA probes, located in the 3 untranslated Inhibitors,Modulators,Libraries region, were generated by in vitro transcription from a PLXND1 PCR product which was flanked by T7 and T3 promoters as described.
Follow ing deparaffinization, 4 m tumor sections were treated with proteinase K at 37 C Inhibitors,Modulators,Libraries for 15 minutes and post fixed in formaldehyde. Non specific binding sites were blocked by incubation with acetic anhydride at room temperature. Tis sues were hybridized with digoxigenin labelled RNA probes at 63 C with 200 ng ml probe. Single stranded non hybridized HTS RNA was degraded with RNAse T1 at 37 C for 30 minutes.