Immunohistochemical analysis staining TNF or IL6 in mouse tissue sections.  Samples are incubated in murine TNF (ab9739) or IL6 primary antibody. Bound primary antibodies were detected using IHC detection kit (ab64261). Immunohistochemistry was used to determine the effect of uridine on synovial expression of TNF and IL6. 

Immunohistochemical analysis of KLF4 and pVHL expression in colon cancer tissues. Sections from colon cancer and adjacent tissues were analyzed by immunostaining against KLF4 and pVHL. Staining without primary antibody served as negative control.

Tissue sections were de-waxed with xylene and rehydrated through gradient ethanol into water. For antigen retrieval, sections were heated in citrate buffer (pH6.0) for 10 min at 95°C in a microwave oven. After cooling to room temperature, the sections were then digested with 0.05% trypsin for 10 min at 37°C. Endogenous peroxidase activity was quenched with 0.3% H2O2 in methanol for 30 min at room temperature. After PBS washes, nonspecific antibody binding was blocked by pre-incubating slides with 10% normal goat non-immune serum at 37° for 30 min. After blotting off the blocking serum, sections were incubated with primary antibody against LKF4 as well as primary antibody against VHL at 4° overnight. After PBS washes again, sections were incubated with biotinylated secondary antibody at 1:200 dilution for 30 min at room temperature. After incubating with Vectastain ABC reagent (Vector Laboratories, Inc., Burlingame, CA) for 30 min at room temperature, the sections were developed with Anti Rabbit HRP/DAB Detection Kit (ab64261). Sections were washed in running tap water and lightly counterstained with hematoxylin, followed by dehydration and coverslip mounting. Negative controls were obtained by omitting the primary antibody.