...The experimental setting presented here is based on an in vitro model using rat vascular smooth muscle cells and the detection of senescence and osteoblastic markers via immunofluorescence and RNAscope™. Co-staining of the senescence marker p21, the osteoblastic marker osteopontin, detection of senescence-associated heterochromatin foci, and senescence-associated β-galactosidase is possible within one test approach requiring fewer cells. The protocol is a fast and reliable evaluation method for multiplexing of calcifying and senescence markers with fluorescence microscopy detection. The experimental setting enables analysis on single cell basis and allows detection of intra-individual variances of cultured cells.