On the 60th day, the mice were anesthetized with 2% Phenobarbital (60 ml/kg, i.p ., and then were sacrificed through cervical dislocation, the livers were immediately collected and were processed for examination using electron microscopy, determination of ROS and lipid oxidation, and analysis of gene expression.
As previously described [ 35], cells were fixed and processed for examination with a JEM 1010 transmission electron microscope (JEOL).
After the designated time point, cells were washed thrice with DMEM followed by the application of 0.5 ml of homogenization buffer (250 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF)), in which cells were gently detached using a cell scraper, lysed, and further processed for examination by a sucrose flotation assay as previously described [ 36].
Following the 7-week duration of the experiment, animals were euthanized and portions of the tumors were processed for histological examination, examination of cell subtypes by FACS analysis of single cell suspensions, and examination of the levels of tumor-associated chemokine proteins by ELISA of tumor homogenates.
After termination, a gross pathological examination was performed, during which the liver and gallbladder, spleen, lungs, and stomach were collected and processed for histological examination.
The testes were processed for histological examination.
Extraabdominal bowels with fibrous peel were processed for histologic examination.
At the end of the experiments, all xenografts were harvested and processed for microscopic examination.
The skin samples were processed for the examination using light microscopy and scanning electron microscopy.
Tumors that arose were harvested and processed for histological examination.
