3.5.3. Sample Collection

At the 21st day of EAC cell inoculation, the mice were anesthetized with thiopental sodium (50 mg/kg). Blood samples were collected from the orbital sinus (retro-orbital plexus). The blood samples were left to clot for 20 min, followed by separation of the serum by centrifugation at 2000× *g* for 15 min. The mice were sacrificed by cervical dislocation, and the tumor discs were separated, weighed, and separated into two portions; one portion of each tumor disc was fixed in 10% neutral buffered formalin for immunohistochemical investigations, while the other portion was kept at −80 ◦C for PCR analysis.

3.5.4. Determination of Endothelial Growth Factor B (VEGF-B) and Tumor Necrosis Factor-α (TNF-α) in the Serum by ELISA

The serum samples were stored at −20 ◦C and used for determination of the levels of VEGF-B and TNF-α by ELISA kits ab213897 and ab181421, respectively (Abcam, Cambridge, UK), according to the manufacturer's instructions. The serum levels of the liver function enzymes ALT and AST were assessed by colorimetric kits AL1031 and AS1061, respectively (Biodiagnostic, Giza, Egypt). Similarly, the serum levels of the kidney markers urea and creatinine were also determined calorimetrically via UR2110 and CR1250, respectively (Biodiagnostic, Giza, Egypt).

3.5.5. Quantitative Real-Time PCR (q RT-PCT) for Assessment of the Expression of Midkine (MDK) in Tumor Tissue

The total RNA was isolated from the tumor tissue by an SV total RNA isolation system (Promega, Madison, WI, USA) according to the manufacturer's instructions. The extracted RNA was stored at −80 ◦C. The concentration and purity of the isolated RNA were measured by a Nanodrop NA-1000 UV/Vis spectrophotomter (Thermo Fisher Scientific Inc., Wilmington, DE, USA). A GoTaq® 1-Step RT-qPCR System (Promega, Madison, WI, USA) and the two primers, 5-GTCAATCACGCCTGTCCTCT-3 (forward) and 5- CAAGTATCAGGGTGGGGAGA-3 (reverse), were used for determination of the MDK expression. *β*-actin was marked as the housekeeping gene and was amplified using two primers: 5-ACGGCCAGGTCATCACTATTG-3 (forward) and 5-CAAGAAGGAAGGCT GGAAAAGA-3 (reverse). The 20-μL reaction mixture of each sample was composed of 4 μL of the RNA template, 1 μL of each of the forward and reverse primers, 0.4 μL of GoScript™ reverse transcriptase (RT) mix for 1-step RT-qPCR, 10 μL of GoTaq® qPCR master mix, 0.31 μL of supplemental CXR reference dye, and 3.29 μL of nuclease-free water. The reaction was carried out in a StepOnePlus™ Real-Time PCR thermal cycler (Applied Biosystems, Waltham, MA, USA). The program was formed of reverse transcription at 37 ◦C for 15 min, inactivation of the reverse transcriptase enzyme, and initial denaturation at 10 min at 95 ◦C, followed by 40 cycles of denaturation at 95 ◦C for 10 s, annealing at 52 ◦C for 30 s, and extension at 72 ◦C for 30 s. The cycle threshold (Ct) of each reaction was recorded, and the ΔCt was calculated against *β*-actin. The fold change of each sample was calculated to be 2−ΔΔCt.

3.5.6. Immunohistochemical Assessment of the Expression of Apoptotic Markers in the Tumor Tissue

The tumor discs were fixed in 10% neutral buffered formalin overnight and then embedded in paraffin. Deparaffinization was performed by adding xylene and ethyl alcohol in decreasing concentrations from 100% to 70%. Antigen retrieval was performed according to the Tris/EDTA buffer (pH = 9) antigen retrieval protocol. The EnVision™ FLEX HRP-labeled high-pH method was used for staining according to the manufacturer's protocol (Dako, Glostrup, Denmark). The primary polyclonal antibodies for p53, Bax, and caspase 3 (Bioss Inc., Woburn, MA, USA) were diluted in PBS (normal phosphate buffered saline) at a ratio of 1:250. Finally, Mayer's hematoxylin was used for counterstaining.

ImageJ was used for the semiquantitative analysis of the immunohistochemical reactions. The images were captured by an optical microscope with a 40× objective (Optika B-352A, OPTICA, Via Rigla, Italy) coupled to a camera (HDCE30C) using its software and

quantified using the ImageJ MacBiophotonics (National Institutes of Health, Bethesda, MD, USA) software package developed by McMaster University (Hamilton, Ontario, Canada). The expressions of p53, Bax, and caspase 3 were all assessed, and the percentages of stained areas were calculated using the color deconvolution plugin.
