Although the reported low abundance of 5mC oxidation derivatives, 5fC and 5caC in some tissues would present significant limitations for a standard immunochemistry protocol, the incorporation of peroxidase-conjugated secondary antibodies allowed the detection of these cytosine modifications in fixed tissues and cells (Figure 2). However, the optimal incubation time with the tyramide solution should be optimized experimentally for each individual batch of tyramide signal amplification kit where a linear relationship between signal intensity and duration of tyramide based signal amplification is observed, for details refer to Almeida et al. 201226. In addition, the signal/background ratio can be significantly enhanced by carrying the washes in a Coplin jar to allow efficient removal of excess antibodies. Following incubation with tyramide solution, it is important to immediately stop the reaction by washing in PBT solution to decrease background staining. It is critical not to allow the sections to dry at any point during the procedure.