DNA methylation and histone modifications are key regulatory mechanisms in cellular differentiation, and are skewed in complex diseases. Therefore, analyzing the higher nuclear organization of methylated DNA in conjunction with relevant cellular components, such as protein biomarkers, may well add cell-by-cell-specific spatial and temporal information to quantitative molecular data for the discovery of stem cell differentiation-related signaling networks and their exploitation in the therapeutic reprogramming of cells. The in situ fluorescent covisualization of methylated DNA (methylated CG dinucleotides = MeC), global DNA (gDNA), and proteins has been challenging, as the immunofluorescence detection of MeC sites requires thorough denaturing of double-stranded DNA for antigen (methylated carbon-5 of cytosine) retrieval. The protocol we present overcomes this obstacle through optimization of cell membrane permeabilization, acid treatment, and intermediate fixation steps to preserve immunostaining of biomarkers and delineate MeC and gDNA, while conserving the captured three-dimensional (3D) structure of the cells; making it suitable for high-resolution confocal microscopy, 3D visualization, and topological analyses of fixed cultured cells as well as fresh and frozen tissue sections.