Immunofluorescence Staining of 5-methylcytosine

Immunofluorescence staining was performed in four-chamber microplates (ThermoFisher Scientific) according to previously established protocols (25 (link), 28 (link), 33 (link)). The primary and secondary antibody set included unconjugated mouse anti-5-methylcytosine monoclonal antibody (AMM99021, Aviva Systems Biology) at 1 mg/ml and Alexa488-linked donkey anti-mouse IgG (A-21202, ThermoFisher Scientific) at 5 mg/ml final concentrations. The cells were subsequently delineated with the cytoplasmic marker Cell Mask Red (ThermoFischer Scientific) and cell nuclei counterstained with DAPI. The specificity/dynamic range of the anti-5mC antibody was tested as previously reported in (29 (link)) (data not shown in here). Formalin-fixed tissue sections at 5 µm thickness were kept floating in 10% formalin at 2−8°C until immunofluorescence staining. Prior to staining, tissues were transferred to microwell plates, washed in PBS at room temperature, then stained as floating tissues using the same protocol that was applied to fixed cells.