Here, we describe a highly sensitive immunochemical method that provides robust and a rapid detection of oxidized form of cytosine in mammalian brain tissue. By incorporating peroxidase-conjugated secondary antibodies coupled with a signal amplification step, this method circumvents the challenges of detecting the very low amounts of 5fC and 5caC. In addition, this technique can be used to co-detect the modified forms of cytosine with lineage specific markers, effectively complementing other approaches in elucidating the biological functions of these epigenetic marks.