Apoptosis and Proliferation Analysis

Ultra-thin sections (5 µm) were cut vertically and mounted on microscope slides. Nuclei were counterstained using Draq5 (BioLegend). Apoptotic cells were labeled using the TUNEL assay (In situ cell death detection kit, TMR red; Merck, Darmstadt, Germany) according to manufacturer’s specifications. Proliferating cells were labeled using an anti-Ki67 monoclonal antibody (primary antibody: rabbit anti-Ki67; Bethyl Laboratories, Montgomery, TX, USA) that was marked using a fluorescently labeled secondary antibody (donkey anti-rabbit IgG Brilliant Violet 421; BioLegend)). Microscopy slides were examined using a Keyence BZ-9000 fluorescence microscope (Keyence, Frankfurt, Germany). Using the software dynamic cell count (BZ-II Analyser, Keyence, Frankfurt, Germany), the ratio of TUNEL positive and negative cells (TUNEL⁺/Draq5⁺ vs. TUNEL^-/Draq5⁺) was determined [43 (link)].