sentence: After washing , wells were blocked with 2 % BSA and probed with anti-5hmC antibody ( Active motif , Cat # 39769 , 1 : 3000 ) at 4 ° C overnight and visualized by HRP-conjugated anti-rabbit secondary antibody ( Santa Cruz , Cat # sc-2004 , 1:10,000 ) and developed by TMB ( 100 µ l / well ; SIGMA , T4444 ) and the color development was stopped by adding 50 µ l of 2 M H SO and measuring optical densities at 450 nm .