Competitive ELISA analysis of 5-mc was performed by coating wells of a 96-well plate with 50 µl per well of 5-mc-BSA diluted in carbonate/bicarbonate buffer, at a concentration of 1 µg/mL overnight at 4℃. Wells of the plate were washed, blocked with 1%BSA blocking buffer, and incubated with 100 µl per well of 5-mc monoclonal antibody at concentration of 1 µg/mL with serial diluted 5-mc and its analogs starting from a concentration of 10,000ng/mL to 0.61ng/mL for 1 hours at room temperature. The plate was washed and incubated with 50 µl per well of an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1/35,000 for one hour at room temperature. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm. This antibody demonstrates high specificity for 5-mc and little or no crossreactivity to it analogs.

Immunohistochemical analysis of paraffin-embedded human colon tissue with Mouse anti-5-methylcytosine (5-mC) antibody (HA610208) at 1/200 dilution. The section was denatured in 2M HCl for 15 minutes at room temperature, then neutralized in 0.1M Tris-HCl (pH 8.3) for 10 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA610208) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.