The protein expression profile of ACE2 in human tissues

Feria Hikmet; Loren Méar; Mathias Uhlén; Cecilia Lindskog

Formalin-fixed, paraffin-embedded (FFPE) tissue blocks from the pathology archives were selected based on normal histology using a hematoxylin-eosin stained tissue section for evaluation. Tissue microarray (TMA) generation, immunohistochemical staining and high-resolution digitalization of stained TMA slides has been described previously 28 . In short, representative 1 mm diameter cores were sampled from FFPE blocks and assembled into an array of normal tissue samples, corresponding to a toal of 144 individuals. TMA blocks were cut in 4 µm sections using a waterfall microtome (Microm H355S, ThermoFisher Scientific, Freemont, CA), placed on SuperFrost Plus slides (ThermoFisher Scientific, Freemont, CA), dried overnight at room temperature (RT), and then baked at 50°C for at least 12 h. Automated immunohistochemistry was performed by using Lab Vision Autostainer 480S Module (ThermoFisher Scientific, Freemont, CA), as described in detail previously 28 . Primary antibodies against human ACE2 were the polyclonal rabbit IgG antibody HPA000288 (Atlas Antibodies AB, Sweden) and monoclonal mouse IgG antibody CAB026174 (cat # MAB933 Human ACE-2 antibody, R&D Systems, Minneapolis, MN). The antibodies were diluted and optimized based on IWGAV criteria for antibody validation 12 . Protocol optimization was performed on a test TMA containing 20 different normal tissues. The stained slides were digitalized with ScanScope AT2 (Leica Aperio, Vista, CA) using a 20x objective. The tissue cores were manually annotated by certified pathologists (Lab SurgPath, Mumbai, India), and curated by a second observer. Annotation parameters included staining intensity, quantity of positive cells and subcellular localization in 78 different normal cell types. All high-resolution images are readily available in version 19.3 of the Human Protein Atlas (https://www.proteinatlas.org).