Serial 3-μm sections from all samples were depara nized and rehydrated through xylenes and serial graded ethanol to water followed by antigen retrieval. These samples then were incubated overnight at 4 °C with TCP1 alpha primary antibody (Abcam Corporation; 1:200). The washed tissue samples were incubated with secondary antibody IgG (Merck Millipore; 1:300) for 30 min at room temperature (RT).
Tissue slices were stained with 3,3'-diaminobenzidine and hematoxylin, and observed under an optical microscope. Finally, all images were analyzed integrated optical density (IOD) to calculate the average IOD /TCP1 positive staining area (µm 2 ) using Image-pro plus software.
Tissue slices were stained with 3,3'-diaminobenzidine and hematoxylin, and observed under an optical microscope. Finally, all images were analyzed integrated optical density (IOD) to calculate the average IOD /TCP1 positive staining area (µm 2 ) using Image-pro plus software.