Indirect ELISA and competitive ELISA results show that this antibody is specific to m5C. Indirect ELISA (blue curve, refer to top X-right Y axis) was performed by coating BSA conjugated m5C at 0.4ng/well followed by blocking with 1% BSA. Serial diluted primary antibody was added to the plates and incubated at 37℃. HRP-goat anti-mouse was used for detection. Competitive ELISA was performed similarly except that different concentration of m5C or its structure analogue compound Cytidine was mixed in 20ng/mL primary antibody (red curve, refer to bottom X-left Y axis).

Total DNA was isolated from HeLa cell line and was dotted to NC membrane at different amount as indicated above the dots. The membrane was blocked with BSA and blotted with 5-methylcytosine antibody 68301-1-Ig at 1:5000 followed by incubation of HRP-goat anti-mouse secondary antibody. Signal was developed by ECL substrate. A parallel dot blot was performed using Mouse IgG2b isotype control Monoclonal antibody 66360-3-Ig at the same dose.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue slide using 68301-1-Ig (5-methylcytosine antibody) at dilution of 1:5000 (under 10x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).

Immunohistochemical analysis of paraffin-embedded mouse testis tissue slide using 68301-1-Ig (5-methylcytosine antibody) at dilution of 1:5000 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).

Immunohistochemical analysis of paraffin-embedded rat brain tissue slide using 68301-1-Ig (5-methylcytosine antibody) at dilution of 1:5000 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).

Immunohistochemical analysis of paraffin-embedded rat testis tissue slide using 68301-1-Ig (5-methylcytosine antibody) at dilution of 1:5000 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).