Flex microscope slides

To test apoptosis in skin cells, tissue sections on immunohistochemistry (IHC) FLEX microscope slides (Dako Denmark, Glosturp, DK) were incubated with a cleaved caspase-3 antibody (#MAB835; RD Systems Donji, Kneginex, HR) at 4 °C overnight in a wet chamber. The next day, the primary antibody was removed, slides were washed in PBS and incubated with Alexa Fluor™ 546 goat anti-rabbit antibody (#PA5-16891; Thermofisher Scientific, Waltham, US) at room temperature for 1 h. After incubation, the secondary antibody was removed, the slides were washed in PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 1 min. After another washing step, slides were mounted (Fluoromount-G, SouthernBiotech, Birmingham, US) covered (Menzel-Gläser Coverslips, Thermofisher Scientific, Waltham, US) and stored at 4 °C. To quantify caspase-3⁺ cells, 4 fields of view (FOV) per slide have been counted using an Olympus AX70 microscope (Olympus, Tokyo, JP). Pictures were obtained via Spot RT3 camera (Visitron Systems GmbH, Puchheim, DE) and MetaMorph software (Molecular Devices, San Jose, US).

Four µm thin sections of the TMA were mounted on Flex microscope slides (Dako) and allowed to dry overnight at room temperature before immunohistochemistry (IHC) processing. For further details, please refer to the supplementary methods.