Hematoxylin and eosin

Formalin-fixed tissues were embedded in paraffin blocks and sectioned (Instrumentation Resource Facility at the University of South Carolina). Livers were stained with hematoxylin and eosin (Biocare, Pacheco, CA). Representative images were taken at 10x (Nikon E600).

HT-29 tumors were dissected at a size of about 1,000 mm³ and directly transferred to formalin. After fixation the tumors were embedded in paraffin and cut in 4 μm-sections. The sections were stained with hematoxylin and eosin (Biocare Medical) using a Tissue-Tek Prisma Plus, Sakura instrument according to the manufacturer’s instructions. Immunostaining with Anti-Ki67 (DAKO, M7240) and Anti-Annexin V (abcam, ab108321, rabbit) was performed after pretreatment with Targen Retireval Solution, Low pH, Dako, K800521-2; antibody dilution 1: 1000, 30 min RT and cMet (Abcam, ab51067), pretreatment – Target Retrieval Solution, High pH, Dako, K800421-2; antibody dilution 1: 100, overnight incubation then detected with Dako EnVision Flex High pH # K801021-2 kit.
Necrotic areas were quantified by estimating the fraction of necrosis in the whole tumor area in five 10× power fields. Mitotic and apoptotic cells (Annexin V positive) were scored in ten 40× power fields. cMet expression scored according to staining intensity (negative -, weak+, moderate++, or strong+++). The proliferation index was quantified by calculating the fraction of Ki67 positive cells in an 0.25 mm² grid in a hot spot area. All histopathologic evaluation was done in a blinded manner by a pathologist.

To evaluate the effect of CDP-choline on cardiac myocytes, 4 × 10⁴ cells were plated onto 96-well plates and incubated in the presence of 0.5, 1, 2.5, 5, 10, 20, 30, 40, 50, 75, and 100 mg/mL of CDP-choline (Ferrer Internacional, S.A., Barcelona, Spain) for 24 and 48 hrs. The number of cells was determined by using the colorimetric MTT assay. For cardioprotection assays, CDP-choline (10, 25, and 50 mg/mL) was added either 1 hr before (preconditioning) or 1 hr after (postconditioning) reperfusion. The number of viable cells was determined after 24 hrs. To assess changes in cell morphology, cells were washed twice with cold PBS and fixed in 100% ethanol overnight at room temperature. The remaining ethanol was discarded, and the cells were rehydrated with double distilled water for 5 min. The cells were stained with hematoxylin and eosin (Biocare Medical, CA, USA). Stained cells were analyzed using a light microscopy Axiostar Plus (Karl Zeiss Microscopy GmbH, Jena, Germany). To evaluate the effect of CDP-choline on oxidative stress-induced death, 5 mM H₂O₂ (Sigma Chemical Company, St. Louis, MO, USA) was added immediately after reperfusion, and the cells were further incubated for 6 hrs.