•The first immunofluorescence microscopy attempt of targeting two different types of proteins on the same cultural heritage micro sample.•A two-step antibody hybridization procedure to analyse two different types of protein binders in real wall paintings.•This procedure allowed us to reduce the costs and operational difficulties, which are normally encountered, when mixing two types of primary antibodies.•Ovalbumin was found in 3 micro samples and casein in 1 micro sample; and none proved positive for both binder types. The aim of the study was to use immunofluorescence microscopy to identify two types of protein binders in 10 polished cross sections of micro samples, which were obtained from real and mock-up wall paintings. A two-step antibody hybridisation procedure was employed, in which the same micro samples were hybridised with anti-ovalbumin and subsequently with anti-casein antibodies and between the hybridisation steps, the sample's cross section was slightly re-polished and cleaned in order to remove all primary and secondary antibody remnants, which remained attached from the first hybridisation step (anti-ovalbumin). This allowed us to reduce the costs and operational difficulties, which are normally encountered, when antibodies, targeting two different proteins, are simultaneously mixed together. To reduce unspecific fluorescence, to amplify the fluorescence of the proteinaceous binders and to construct 3D surface topography models, apart from widefield fluorescence, laser-scanning confocal microscopy was performed. In parallel, FTIR spectroscopy analysis, of finding proteinaceous materials in micro sample cross sections, was also conducted. Results show that our two-step hybridisation procedure allowed for an accurate localisation of both types of proteinaceous binders without any interference from the first hybridisation step. Three micro samples proved positive for ovalbumin and one for casein and none of these proved positive for both analysed binder types. For eight micro samples, FTIR spectroscopy results completely matched those of immunofluorescence microscopy. According to our knowledge, this was the first immunofluorescence microscopy attempt of targeting two different types of proteins on the same cultural heritage micro sample.