Histology & fluorescence microscopy

For the in situ GFP-LC3 fluorescence analysis, mice were euthanized by carbon dioxide asphyxiation, then immediately perfused through the left ventricle first with cold dulbecco’s phosphate-buffered saline (DPBS; ThermoFisher, 21600–044), followed by 4% paraformaldehyde (Sigma, 6148) in DPBS to fix the tissue. Samples were collected and further fixed with the same fixative for 4 h, followed by treatment with 15% sucrose (Sigma, S9378) in DPBS for 4 h at room temperature and then with 30% sucrose solution overnight at 4°C. Tissue samples were embedded in optimal cutting temperature (OCT) medium (Tissue-Tek, 4583) and stored at -80°C. 10 µm sections were investigated for the presence of GFP-LC3 puncta using a confocal laser scanning microscope (LSM880 and LSM700, Zeiss, Germany) in super resolution mode. Channels were acquired separately. For the visualization of autophagosomes at the corneum high laser power and multiple averaging was applied. Quantification of epidermal keratinocytes undergoing CDA was performed as following: cells with either a high number of autophagosomes or a high concentration of GFP-LC3 at the nucleus were counted in three independent SR-LSM pictures of three biological samples/genotype each and displayed as the percentage of CDA cells relative to the total number of cells (determined by counting the nuclei). Conventional fluorescence was performed on an AxioImager Z2 (Zeiss, Germany). The primary antibody, rabbit anti-IVL/involucrin (Covance, PRB-140C) was used in a 4°C overnight incubation at a 1:500 dilution DPBS/2% BSA/10% normal horse serum (Vector Labs, S-2000), goat anti-CTSD 1:1000 (R&D Systems, AF1029) in a concentration 5 µg/ml. Hoechst 33258 (Life Technologies, H3569) or DAPI (4′,6-diamidino-2-phenylindole, Sigma, D9564) 1 ng/ml was used to label the nuclei. The secondary antibodies, donkey anti-rabbit Alexa Fluor 647 (Life Technologies, A-31573) or donkey anti-goat Alexa Fluor 647 (Life Technologies, A-21447), respectively, were used at a 1:500 dilution. For TUNEL staining, the in situ Cell Death Detection Kit TMR red Roche (Sigma, 11684795910) was used. H&E staining of sections from paraffin-embedded tissue was performed according to standard protocols.