Fig 2: ACE2 protein expression within human vascular endothelial cells and the lung.a Representative double immunofluorescence staining of ACE2 and endothelial cell marker CD31 in the blood vessels of human nasal turbinates using six different anti-ACE2 antibodies and anti-CD31. b Double immunofluorescence staining of ACE2 and type II pneumocyte marker mucin 1 (MUC1) in the human lung using six different anti-ACE2 antibodies and anti-MUC1. Abcam ab15348 clone yielded the most robust staining of pneumocytes, while the other clones showed negligible or less specific membrane staining. Scale bars: 20 μm (top) and 5 μm (bottom).

Fig 3: Analysis of human primary cells, infected with the VOC 202012/01 lineage B.1.1.7 variant in the absence or presence of D3, by immunofluorescence assays and RT-PCR. (a) The cells untreated or infected with the VOC 202012/01 variant of SARS-CoV-2, in the absence or presence of D3 for 72 h at 37 °C, were fixed, washed and permeabilised, as described in the Methods. After blocking, the slides were incubated with the relevant primary antibodies overnight at 4 °C: anti-ACE2 (1:100; ab15348; Abcam) or anti-SARS-CoV-2 Nucleoprotein (N) Antibody (1:100; No. 35-579, ProSci), followed by the relevant secondary anti-mouse Alexa Fluor 488 (1:200; ab150113; Abcam) and anti-Rabbit Alexa Fluor 546 (1:200; A-11035; ThermoFisher), respectively. DNA was stained with DAPI (1:5000; #62254; Thermo Fisher). Microscopy images were obtained with the Elyra 7 platform (Zeiss) with the optical Lattice SIM technology, using the 63 × oil immersion objective. (b) Quantification of the mean fluorescence intensity was performed by using the ZEN software (Zeiss, black edition). (c) Cell extracts were analyzed by RT-PCR for measuring the expression of N1 viral gene and the levels of the indicated cytokines. In parallel, the extract of uninfected cells was used as negative control.

Fig 4: Immunocytochemistry analysis of ACE2 on ejaculated human sperm cells. Panel (A) shows the location of the C-terminal domain of ACE2 recognized by anti-ACE2-1 (Abcam, ab15348), shared between both isoforms; Panel (B) shows the fluorescence pattern after immunocytochemistry analysis with anti-ACE2-2 (Novus, NBP2-67692), recognizing the full-length ACE2 only. DAPI was used to stain the nuclei. Top: two-laser image (anti-ACE2 antibody + DAPI); middle: sperm cells stained only with DAPI; bottom: sperm cells illustrating only ACE2 fluorescence patterns (scale bar = 5 µm). Negative controls for immunocytochemistry assays are shown in Supplementary Figure S1.

Fig 5: Immunohistochemical analysis of ACE2 protein localization across human tissues using multiple anti-ACE2 antibodies.Representative images of human tissues on a tissue microarray (TMA) stained by chromogenic immunohistochemistry using antibodies targeting the ACE2 protein (brown) and counterstained with hematoxylin (blue). Highest ACE2 expression was observed in the villi of the intestinal tract (jejunum), renal tubules, testis, and glandular cells in the seminal vesicle. Minimal to no/non-specific staining can be seen in the heart, stomach, spleen, skin, and liver. Staining of lung pneumocytes was observed using Abcam ab15348, and less specifically with Sigma HPA000288 (Fig. 2b; Supplementary Table 1). Scale bars: 100 μm.