Nitroimidazo oxazines were found to be superior to the CGI 17341 compounds due

The slides were washed with PBS Lapatinib three times and then incubated for 1 hour at room temperature with goat antirat Cy5 secondary antibody. Nuclear counterstain with Sytox green was applied for 10 minutes, and a mounting medium was placed on each sample, which were then covered with a glass coverslip. Endothelial cells were identified by green fluorescence, whereas EGFR or pEGFR positive cells were identified by red fluorescence. The presence of EGFR or pEGFR on endothelial cells was detected by colocalization of red and green fluorescence, which appeared yellow. Immunohistochemical Determination of Ki 67 Antigen, CD31, and TUNEL Paraffin embedded tissues were used for immunohistochemical staining for Ki 67 as previously described. Ki 67 labeling index was determined by light microscopy at the site of the greatest number of Ki 67positive cells. The representative areas were determined by scanning tumor sections using low power. For Ki 67 LI, the number of positive cells among approximately 1000 tumor cells was calculated as a percentage. Lymphatic system Frozen tissues were used for quantifying mean vessel density. Frozen sections were fixed in cold acetone, and immunohistochemical procedures were done as described previously. For the quantification of MVD, 10 random 0. 159 mm2 fields at a magnification of 100 were captured, and CD31 positive cells were quantified according to a method described previously. Analysis of apoptotic cells was done by using a commercially available TUNEL kit. To quantify the apoptotic index, the TUNEL positive cells were counted in 10 random 0. 159 mm2 fields at a magnification of 100. Double Immunofluorescence Staining for CD31 and TUNEL Frozen sections of cecal tumors were used for assay. Specimens were cut into 4 um sections, mounted on positively charged slides, and stored at 80 C. Slides were JZL184 fixed in cold acetone for 10 minutes, placed in a light shielded humidified chamber, incubated with protein blocking solution for 20 minutes at room temperature, and incubated overnight at 4 C with primary antibody against CD31. The slides were washed with PBS three times and then incubated for 1 hour at room temperature with goat antirat Cy3 secondary antibody. Then, TUNEL assay was done by using a commercially available TUNEL kit. Nuclear counterstain with Sytox green was applied for 10 minutes, and slides were covered with a glass coverslip as described in the above paragraphs. TUNEL positive apoptotic cells were detected by localized green fluorescence within the cell nuclei, and endothelial cells were identified by red fluorescence. Apoptotic endothelial cells were detected by colocalization of red and green fluorescence, which appeared yellow, within the nuclei. The total number of apoptotic cells was quantified in 10 randomly selected microscopic fields and expressed as the ratio of apoptotic endothelial cells to the total number of endothelial.