In this system, the abdominal normally cavity of the mouse was divided into 4 regions region I, subdiaphragm. region II, the liver, spleen, stom ach, and affiliated ligaments. region III, the Inhibitors,Modulators,Libraries small intestine, colon, mesenterium, and abdominal wall. and region IV, the pelvic cavity, urogenital system, and rectum. The detailed scoring criteria were modified from a similar reporting system on a rat peritoneal carcin omatosis model and set forth in our previous re port. Immunohistochemistry study Tumor tissues obtained from animals of 3 groups were subjected to immunohistochemistry to detect Inhibitors,Modulators,Libraries the ex pressions of Cat B, Ki 67, CD34, VEGF, E cadherin and D2 40, according to our previously reported procedures. The primary antibodies for Cat B, Ki 67, CD34, VEGF, E cadherin and D2 40 were prepared and incubated with the slides for 2 h in a moist chamber.
After a new cycle of washes, the slides were again placed in a moist chamber for 30 minute incubation Inhibitors,Modulators,Libraries with a biotinylated secondary antibody and biotin peroxidase complex. The color of immunoperoxidase reaction was achieved by immersion for 5 min in a solution con taining the DAB chromogen and counterstained with hema toxylin for 2 min. The slides were observed under the microscope. For the evaluation of IHC results, positive cells were stained brownish granules in the cell membrane, cyto plasm or nucleus. In all cases, cytoplasmic Cat B expression was scaled as moderate and strong expres sion. Ki 67 expressed in the nucleus. VEGF positive cells were stained both in the nucleus and cytoplasm. The expression of E cadherin mainly existed in cell membrane and cytoplasm.
CD34 and D2 40 positive cells were stained in cytoplasm. Ten fields in each slide were selected randomly and observed at a magni fication of200. The expression of Ki 67 was evalu ated according to positive rate. The positive expression of CD34 and D2 40 was evaluated according to microvessel density and lymphatic Inhibitors,Modulators,Libraries microvessel density. Western blotting study Fresh tumor tissues in RIPA lysis buffer containing 1 ug ml PMSF, 1Cocktail, were manually homogenized on ice using a glass homogenizer, then centrifuged at 3000 g for 10 min to remove cellular and nuclear debris. The protein concentration was determined using a BCA Assay kit.
To determine the expressions of p ERK12, ERK12, Bcl 2, caspase 3, and B actin using western blotting, 100 ug total proteins were separated by SDS poly acrylamide gel electrophoresis and then transferred overnight onto PVDF membranes, which were blocked with 5% skimmed milk in 0. 01 M phosphate buffer solution containing Inhibitors,Modulators,Libraries 0. 05% Tween. Next, they were immunoblotted with a rabbit anti human p ERK, sellekchem rabbit anti human ERK, rabbit anti human Bcl 2, rabbit anti human caspase 3, mouse anti human caspase 9, and rabbit anti human B actin for 3 h.