Image pro plus 4.5 system

The protein expression of PI3K, p-Akt and HIF-1α were analyzed by immunohistochemical staining. The anti-PI3K, p-Akt and HIF-1α antibodies were used at 1:100 dilutions. Endogenous peroxidase was inhibited by incubation with freshly prepared 3% hydrogen peroxide with 0.1% sodium azide. Non-specific staining was blocked with 0.5% casein and 5% normal serum. The tissues were incubated with biotinylated antibodies and horseradish peroxidase (Cell Signaling Technology, Inc.). Staining was developed with diaminobenzidine substrate and sections were counterstained with hematoxylin and eosin. Normal serum or phosphate-buffered saline (PBS) replaced the antibodies in negative controls. The images were analyzed with Image-Pro Plus 4.5 System (Media Cybernetics, Inc., Rockville, MD, USA). The total optical density value and area of intracellular fluorescence for each section was measured.

The mice were killed under anaesthetization and transcardially perfused with 200 mL normal saline and then with 400 mL 4% paraformaldehyde solution. The brains were removed and fixed in the same fixation fluid for another 24 hour at 4°C. For each primary antibody, five consecutive sections from each brain were used. The immunoreaction was detected with avidin horseradish peroxidase‐labelled antibodies and visualized with the diaminobenzidine tetrachloride system. The immunofluorescence was measured by the fluorescent secondary antibody (1:200, A‐11034 Invitrogen) for 1 hour. The images were observed with a microscope. For each experimental group, we measured 10 random images (6 slices from 3 mice). The Image‐Pro Plus 4.5 system (Media Cybernetics Inc, Rockville, USA) was used to estimate the intensity of the immunohistochemical reaction.³⁷