The sections were then washed with PBS at room temperature 3 times for 1 min each, dried, and incubated http://www.selleckchem.com/products/MG132.html with anti-streptavidin-peroxidase (ApopTag Plus peroxidase kit, Millipore, Billerica, MA, United States) at room temperature for 30 min. The sections were then washed with PBS 4 times for 2 min to determine the visibility of the TUNEL reaction before being stained with diaminobenzidine (DAB) (DAB-PLUS kit; Invitrogen, Carlsbad, CA, United States). After washing with distilled water, ground staining was performed using methyl green. After three changes of the searing process with xylene for 20 min, it was closed with Entella. Tissue homogenization and measurement of tissue serum TNF alpha The tissue samples obtained from the ileum were introduced into 2 mL microcentrifuge tubes and stored at -80��C until the day of the study.

These tissues were then removed and warmed to 4 ��C. Next, 60-80 mg pieces were obtained from these samples and placed into a tube containing 5-mm-diameter stainless steel beads and a phosphate buffer with a 1:7 ratio (pH 7.2). Microcentrifuge tubes were introduced into a pre-chilled TissueLyser LT device and replaced into a TissueLyser (Qiagen-Germany) tissue homogenization device. Next, an enzyme-linked immunosorbent assay (ELISA) was performed on tissue supernatants, and serum was obtained via centrifugation for the identification of TNF alpha in accordance with the manufacturer��s recommendations (Invitrogen Rat TNF-alpha, Carlsbad, CA, United States). Finally, the ELISA plates were spectrophotometrically evaluated at 450 nm (Biotech Synergy HT; Winooski, VT, United States).

PDGFR alpha and beta levels PDGFR alpha and beta levels were assessed through staining scores and compared among the groups by immunohistochemistry. For immunohistochemical staining, 2-3 micron sections were stored overnight in an incubator at 40 ��C. The following day, the sections were washed with xylene, a descending alcohol series, and distilled water for 20 min. They were then boiled for 20 min in EDTA solution at pH 8. Next, they were stored in DakoFlex peroxidase solution for 5 min and washed again with Tris-buffered saline. A primary antibody was then applied. PDGFR alpha in a 1:100 dilution (NOVUS Biologicals, NBP1-19 423, Littleton, CO, United States) and PDGFR beta in a 1:50 dilution (NOVUS Biologicals, NBP1-19 473; Littleton, CO, United States) were stored for 30 min, washed with Tris buffer, stored in DakoFlex HRP solution for 20 min, washed with Tris buffer again, and stored in DakoFlex DAB for 7 min.

The samples were again washed with Tris-buffered saline, kept under tap water for 5 min, stained with Mayer��s hematoxylin solution for 10 min, washed with tap water for 1 min, rinsed Anacetrapib in an alcohol series, and cleaned with xylene for 5-10 min. PDGFR alpha and beta positivity was determined according to a devised scoring system.