Immune co-labeling of P-gp/caveolin-1 Tumor sections were first permeabilized in 0.01 M PBS containing 0.1% Triton X-100 (PBST) for 3 × 5 min followed by blocking in 10% goat serum in 0.01 M PBST. Antibodies were diluted in 3% bovine serum albumin in 0.01 M PBST. The anti-caveolin-1 antibody was used at concentrations between 0.2-5 mg/ml, and the anti-P-gp antibody was used at a concentration of 5-10 mg/ml. Prior to Gemcitabine application to the tissue, the primary antibody was pre-incubated with the blocking peptide at 37°C for 3 h (caveolin-1, Santa Cruz Biotechnology, Santa Cruz, CA) or overnight at
4°C (P-gp). After thorough washing steps using PBS, sections were incubated for 1 h at room temperature with 5 mg/ml of each secondary antibody. Following washing with PBS, the sections were put on coverslips. The sections were viewed under a Leica TCS-SP2 confocal laser BIIB057 molecular weight scanning microscope
(Leica Instruments Co., Ltd. German) using ×40 and ×63 oil-immersion objective lenses with either ×1 or ×2 zoom factors. On the double-immunolabeled sections, a sequential scan procedure was applied during image acquisition of the two fluorophores, Alexa Fluor 488 (excitation at 488 nm and detection range 500-535 nm; green fluorescence) and Alexa Fluor 568 (excitation at 568 nm and detection range 580-620 nm; red fluorescence). Confocal images were taken at 0.5-μm intervals through the z-axis of the section, and a total depth of 15-20 KU55933 nmr μm was covered. Images from individual optical sections and multiple serial optical sections were analyzed, recorded digitally and stored as TIFF Vildagliptin files in Adobe Photoshop software. All the investigation procedures on human tissues were carried out in accordance with the local institutional ethical committee policies. This study were approved by the relevant regulatory committee of the Chongqing Medical University. Statistical analysis Statistical data were analyzed using the SPSS 10.03 software (SPSS, Chicago,
IL, USA). The rank sum test was used to examine the differential expression of the 5 multidrug resistance proteins in brain tumors. Fisher definite probability methods were used to examine the differences between high grade and low grade tumors. Results were considered statistically significant at P < 0.05. Results Expression of 5 multidrug resistance proteins in brain tumors In our study, we find the expression of multidrug resistance proteins in three different positions, including tumor cells, capillary vessels and interstitial cells. In tumor cells, the strongly positive rate of P-gp, Topo II, GST-π, LRP and MRP were 26.67%, 23.33%,16.67%,3.33% and 3.33%, respectively (Tab 1). This difference was statistically significant (Rank sum test, P < 0.
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