This selective one-front localization suggests that P-gp plays a barrier protective role by extruding cytotoxic substances and drugs from the endothelial cells back into the bloodstream . Another view is that the site of expression of P-gp is also in perivascular astrocytes in the human brain [9, 10]. Moreover, recently studies have shown that P-gp is localized to caveolae and co-immunoprecipitates with caveolin-1 , an integral protein of the caveolae frame, suggesting that the two proteins might physically interact. The purpose of the present study was see more to examine the mechanisms of multidrug resistance of brain
tumors and the localization of P-gp in pediatric brain tumors. This in situ study was carried out on tumor tissues by immunohistochemistry using a monoclonal antibody against P-gp. In addition, double immunolabeling was carried out with antibodies TGF-beta inhibitor to P-gp and caveolin-1 by immunofluorescence laser scanning confocal microscopy to ascertain whether there is any association between these molecules in the microvessels of brain tumors. Materials and methods Materials This study included 30 samples of pediatric brain tumor tissues, including 19 astrocytomas, 8 ependymomas, 3 medulloblastomas. The patients were 20 boys and 10 girls ranging between
6 months and 12 years (median 7.6 years) who were undergoing tumor resection without chemotherapy for high grade (III-IV) tumors (10 cases) and low grade (I-II) tumors (20 cases), according to the grade of Malignancy of Brain Tumor in WHO in 2000 . Five brain tissue samples from autopsies (patients died due to cardiovascular Protein Tyrosine Kinase inhibitor disease) were used as controls. Immunohistochemistry Paraffin sections were first rehydrated, and then rehydrated sections were incubated with a 1:200 dilution of rabbit anti-human primary antibody against P-gp (Santa Cruz Biotechnology, Santa Cruz, CA), LRP (ABCOM Information Systems Pvt. Ltd, USA), MRP (Maixin Bio, Fuzhou, China), GST-π (Maixin
Bio, Fuzhou, China), Topo II (ABCOM Information Systems Pvt. Ltd, USA), S-100 (Santa Cruz Biotechnology, Santa Cruz, CA) or control IgG (1:1000) overnight at 4°C. The tissue sections were washed in PBS and then incubated Phosphatidylinositol diacylglycerol-lyase with a 1:100 dilution of biotinylated secondary sheep anti-rabbit or goat anti-rabbit IgG (Jingmei BioTech, Shenzhen, China). After washing with PBS, tissue sections were incubated with an avidin-biotin complex and developed in 0.075% (w:v) 3,3 diaminobenzidine (DAB). After lightly counterstaining with haematoxylin, the sections were dehydrated. P-gp, MRP, LRP, GST-π are expressed in the cell membrane and or cytoplasm, and Topo-II is expressed in the nucleus. A positive reaction is colored brown. The intensity of immunostaining around the stent struts was scored as follows: 0, no staining; 1, minor staining only; 2, moderate staining; and 3, heavy staining. Intensities of 2 and 3 were considered strongly positive and indicate that drug resistance would be induced by the resistance protein.