Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control ( n = 3). b Transepithelial electrical resistance (TEER in Ω cm 2 ) between the apical and basal poles was measured at each time point ( n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a , b are the means ± s.d. of three independent biological replicates. Source data a – d are provided as a Source Data file.

Article Snippet: ACE2 , Bioss (bs-1004R), rabbit polyclonal (1:100).

Techniques: Infection, Marker, Staining

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: Source of antibodies and dyes with work concentration for immunofluorescence.

Article Snippet: ACE2 , Bioss (bs-1004R), rabbit polyclonal (1:100).

Techniques: Concentration Assay, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet: Neutralization of SARS-CoV-2 infection and RBD binding to ACE2 (A) Indicated hmAbs were incubated with live SARS-CoV-2 (100 PFU/well) 1 h before (pre-treatment) or 1 h after (post-treatment) addition to Vero E6 cells. hmAbs were tested in quadruplicate cultures and NT 50 and upper 95% confidence interval (CI) indicated. (B) Representative titration curve of 1212C2 hmAb presented, mean and standard error presented. (C) Binding of indicated hmAb to SARS-CoV-2 or mock-infected Vero E6 cells measured by immunofluorescence; scale bar, 100 μm. (D) Indicated hmAb was incubated as single replicate with recombinant biotinylated RBD protein before incubation with HEK293-ACE2 cells measured by flow cytometry. Plot gated on 7-aminoactinomycin (7AAD)-ACE2 + cells.

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Neutralization, Infection, Binding Assay, Incubation, Titration, Immunofluorescence, Recombinant, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet: SPR epitope mapping (A) Representative sensorgram from the SPR competition assays used to subset hmAbs into distinct RBD binding epitopes. For each assay, a series of hmAbs were sequentially injected over immobilized SARS-CoV-2 RBD. In this example, 1212C2 was injected first, followed by a second injection of 1212C2, 2 injections of 1206D1, and the last injection was CR3022. (B) Summary of all epitope mapping data, in which each block (first experiment from A in the red box) with a bold hmAb at the top represents a different experiment (10 experiments total). The bold hmAb is the “first” hmAb injected. The percentage of binding (100 = 100% binding and 0 = 0% binding) of subsequent hmAbs was recorded. mAbs were considered to have a different epitope (denoted by a distinct color) if they exhibited binding levels >30% in the presence of other mAbs. Thus, in the first experiment, CR3022 is defined as a new epitope (cyan), distinct from 1212C2. (C) Schematic diagram of NmAb RBD epitopes defined in the mapping experiment. Five major epitopes (A–E) were identified, where the E epitope overlaps with control mAb CR3022 (cyan, epitope F). Four of the 5 epitopes (A–D) are located within the ACE2 binding site (purple), and all of the NmAbs are blocked by the 1212C2 epitope A (yellow). NmAbs with epitopes similar to B (orange) and C (green) are defined as B’ (light orange) and C’ (light green), respectively. The 1212C2 epitope A (yellow) blocks the binding of all NmAbs, with the exception of 1215B11, which occupies epitope E. Epitopes B and C are also blocked by epitope A NmAbs, but exhibit limited competition with each other.

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Binding Assay, Injection, Blocking Assay, Control

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet:

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Synthesized, Expressing, Virus, Recombinant, Plasmid Preparation, Binding Assay, Saline, cDNA Synthesis, Transfection, Gel Extraction, Lysis, Luciferase, Software

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control ( n = 3). b Transepithelial electrical resistance (TEER in Ω cm 2 ) between the apical and basal poles was measured at each time point ( n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a , b are the means ± s.d. of three independent biological replicates. Source data a – d are provided as a Source Data file.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Infection, Marker, Staining

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: Source of antibodies and dyes with work concentration for immunofluorescence.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Concentration Assay, Immunofluorescence

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: A) HeLa cells were transduced with ACE2 lentivirus to generate stable cell lines. Mock and ACE2 transduced cells were lysed and immunoblotted for ACE2. Tubulin was used as a loading control. (B) HeLa WT +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green) and DAPI (blue). (C) HeLa WT +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green), tetherin (red) and DAPI (blue). Uninfected cells shown with asterisk. (D) Electron micrographs showing plasma membrane associated SARS-CoV-2 virions and virus filled intracellular organelles. SARS-CoV-2 infected HeLa WT +ACE2 cells (MOI 0.5) were fixed at 24 hpi and processed for TEM. Left micrograph – plasma membrane-associated virus, middle micrograph – virus-filled tubulovesicular compartments are directed towards the plasma membrane, right micrograph – virions within DMVs. (E) Surface immunogold electron microscopy of SARS-CoV-2 infected HeLa WT +ACE2 cells. Cells were infected with SARS-CoV-2 (MOI 0.5), fixed at 24 hpi and immunogold labelled with antibodies against tetherin. (F) As (E) but labelled with antibodies against SARS-CoV-2 spike.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Transduction, Stable Transfection, Control, Infection, Staining, Clinical Proteomics, Membrane, Virus, Electron Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: (A) A549 cells were transduced with ACE2 lentivirus to generate stable cell lines. Mock and ACE2 transduced cells were lysed and immunoblotted for ACE2. Tubulin served as a loading control. (B) A549+ACE2 cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green) and DAPI (blue). Uninfected cells shown with asterisk (C) A549+ACE2 cells were treated with IFNα (1000 U/ml, 24 hours) to upregulate tetherin expression. Cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green), tetherin (red) and DAPI (blue). Uninfected cells shown with asterisk. (D) A549+ACE2 cells were treated with IFNα (1000 U/ml) and infected with SARS-CoV-2 (MOI 0.5), fixed at 24 hpi and processed for TEM. Infected cells display very few virions on their plasma membrane (left inset) but significant DMV formation (right inset). (E) Electron micrograph of the perinuclear region of A549+ACE2 mock infected cells. Zoomed area shows typical Golgi morphology. (F) Electron microscopy of the perinuclear region of SARS-CoV-2 infected A549+ACE2 cells. Cells were infected at an MOI of 0.5 and fixed at 24 hpi. Zoomed area shows membrane rearrangements and DMVs. (G) T84 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi and stained for spike (green) and tetherin (red). (H) T84 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi and processed for TEM. Tethered virions were frequently present at the plasma membrane.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Transduction, Stable Transfection, Control, Infection, Staining, Expressing, Clinical Proteomics, Membrane, Electron Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: (A) Lentiviral ACE2 was used to generate stable HeLa Bst2KO +ACE2 cells, and ACE2 expression was verified by Western blotting. GAPDH served as a loading control. (B) HeLa WT +ACE2 and HeLa Bst2KO +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi. Cells were stained for spike (green) to demonstrate infection with SARS-CoV-2, and tetherin (red). (C) High MOI viral growth curves were performed by infecting HeLa WT +ACE2 and HeLa Bst2KO +ACE2 cells with SARS-CoV-2 at MOI (5). Titres were measured by plaque assays. (D) Low MOI viral growth curves were performed by infecting HeLa WT +ACE2 and HeLa Bst2KO +ACE2 cells with SARS-CoV-2 at MOI (1). Titres were measured by plaque assays.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Expressing, Western Blot, Control, Infection, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: (A) Schematic diagram to illustrate the domain organization of SARS-CoV-1 ORF7a and SARS-CoV-2 ORF7a. SP = signal peptide. TM = transmembrane domain. Expanded region below shows regions flanking the transmembrane domain with amino acid differences (*) and deletions (-) between SARS-CoV-1 ORF7a and SARS-CoV-2 ORF7a. (B) Representative confocal immunofluorescence images of HeLa cells transiently transfected with SARS-CoV-1 ORF7a-FLAG or SARS-CoV-2 ORF7a-FLAG. SARS-CoV-1 ORF7a-FLAG predominately colocalizes with TGN46 (red), while SARS-CoV-2 ORF7a-FLAG shows additional staining outside that colocalizing with TGN46 (arrowheads). (C) Manders’ coefficients were calculated to measure the ORF7a-FLAG overlap with TGN46. n = 3 independent experiments. Means of each independent experiment are plotted. Two-tailed, unpaired t-tests were performed. *** p < 0.001. (D) SARS-CoV-2 infected HeLa WT +ACE2 cells display fragmentation of Golgi markers. HeLa WT +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi. Infected cells were identified by spike staining (green) and cells were costained with Golgi markers TGN46 (top) and ZFPL1 (below). Areas of TGN46 and ZFPL1 are enlarged (right) to highlight Golgi fragmentation in SARS-CoV-2 infected cells.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Immunofluorescence, Transfection, Staining, Two Tailed Test, Infection

Journal: bioRxiv

Article Title: ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

doi: 10.1101/2020.08.31.270736

Figure Lengend Snippet: (A) Bar graph showing the percentage of cells with detectable ACE2 in islets from pancreata of donors with (n=2,705 cells) and without type 2 diabetes (n=12,185 cells). (B) Violin plot showing the distribution of ACE2 normalized expression in islet cells from pancreata of non-diabetic donors. (C) Bar graph showing the percentage of cells with detectable TMPRSS2 in islets isolated from pancreata of donors with (n=2,705 cells) and without type 2 diabetes (n=12,185 cells); * P <0.05, paired t-test for indicated comparisons. (D) Violin plot showing the distribution of TMPRSS2 normalized expression in islets cells from pancreata of non-diabetic donors; ***adjusted P <0.001, Wilcoxon rank sum tests. (E) Representative images of smFISH for ACE2 and TMPRSS2 mRNA in human pancreatic tissue sections counter stained for insulin (magenta). Inset highlights mRNA distribution in pancreatic ducts; scale bar: 20µm. (F) Representative smFISH images showing the presence of ACE2 and TMPRSS2 in CD34 positive cells in human pancreatic tissue sections; scale bar: 10µm. (G) Representative images of smFISH for ACE2 and TMPRSS2 mRNA in human pancreatic tissue sections counter stained for insulin (magenta). Inset highlights distribution in the endocrine pancreas; scale bar: 20µm. See also Table S1, Fig. S1, Table S2 , and Fig. S2A .

Article Snippet: For single-stained kidney, duodenum, and pancreas tissue sections ( and Fig. S2D ) and for SARS-CoV-2 NP single stained lung and pancreas sections ( and Fig. S4 ), slides were incubated overnight at 4°C with one of four primary antibodies against ACE2 (rabbit monoclonal anti-ACE2, 1:200 dilution (Abcam); rabbit polyclonal anti-ACE2, 1:2,000 dilution (Abcam); mouse monoclonal anti-human ACE2, 1:100 dilution, (R&D Systems); goat polyclonal anti-ACE2, 1:100 dilution, (R&D Systems)) or a primary antibody against SARS-CoV-2 NP (mouse monoclonal anti-SARS-CoV-2 NP, 1:50 dilution, Invitrogen).

Techniques: Expressing, Isolation, Staining

Journal: bioRxiv

Article Title: ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

doi: 10.1101/2020.08.31.270736

Figure Lengend Snippet: (A) ACE2 protein structure illustrating the location of respective antibody directed antigen sites: SP, signal peptide; TM, transmembrane domain; CD, cytoplasmic domain. (B) Immunoblot analysis of four commercially available ACE2 antibodies using total pancreas lysates from three control organ donors (p1-p3) with accompanying Actin labeling. (C) Representative IHC images of human pancreas tissue sections stained for ACE2 using four commercially available ACE2 antibodies. Scale bars: 200 µm. Abbreviations: i, islet; d, duct; mv, microvasculature. See also Table S2 and Fig. S2B-D .

Article Snippet: For single-stained kidney, duodenum, and pancreas tissue sections ( and Fig. S2D ) and for SARS-CoV-2 NP single stained lung and pancreas sections ( and Fig. S4 ), slides were incubated overnight at 4°C with one of four primary antibodies against ACE2 (rabbit monoclonal anti-ACE2, 1:200 dilution (Abcam); rabbit polyclonal anti-ACE2, 1:2,000 dilution (Abcam); mouse monoclonal anti-human ACE2, 1:100 dilution, (R&D Systems); goat polyclonal anti-ACE2, 1:100 dilution, (R&D Systems)) or a primary antibody against SARS-CoV-2 NP (mouse monoclonal anti-SARS-CoV-2 NP, 1:50 dilution, Invitrogen).

Techniques: Western Blot, Control, Labeling, Staining

Journal: bioRxiv

Article Title: ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

doi: 10.1101/2020.08.31.270736

Figure Lengend Snippet: (A) Scheme of the experimental setup illustrating human pancreatic tissue processing, whole stained slide imaging, and machine learning algorithm application for the generation of ACE2 protein expression data. (B) Quantification of ACE2 protein expression in the pancreas of control organ donors with ages ranging from birth to 72 years shows progressive developmental changes. Data are presented as mean±SD with analysis by one-way ANOVA and Tukey’s post hoc test for multiple comparisons. (C) Representative confocal images of ACE2 protein expression in pancreatic ducts of control donors across different age groups. Scale bars (left to right): 100µm, 200µm, 200µm, 300µm, 300µm, 300µm. (D) Representative immunofluorescence images showing lack of ACE2 protein expression in pancreatic blood vessels from control donors across different age groups. Scale bars (left to right): 100µm, 200µm, 200µm, 200µm, 200µm, 100µm. (E) Representative immunofluorescence images show ACE2 protein expression in pancreatic microvasculature from control donors across different age groups. Scale bars: 100µm. (F) Representative immunofluorescence images of pancreatic islets showing ACE2 protein expression restricted to the islet’s microvasculature in pancreata from control organ donors across different age groups. Scale bars: 50µm. See also Table S2 and Fig. S3 .

Article Snippet: For single-stained kidney, duodenum, and pancreas tissue sections ( and Fig. S2D ) and for SARS-CoV-2 NP single stained lung and pancreas sections ( and Fig. S4 ), slides were incubated overnight at 4°C with one of four primary antibodies against ACE2 (rabbit monoclonal anti-ACE2, 1:200 dilution (Abcam); rabbit polyclonal anti-ACE2, 1:2,000 dilution (Abcam); mouse monoclonal anti-human ACE2, 1:100 dilution, (R&D Systems); goat polyclonal anti-ACE2, 1:100 dilution, (R&D Systems)) or a primary antibody against SARS-CoV-2 NP (mouse monoclonal anti-SARS-CoV-2 NP, 1:50 dilution, Invitrogen).

Techniques: Staining, Imaging, Expressing, Control, Immunofluorescence

Journal: bioRxiv

Article Title: ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

doi: 10.1101/2020.08.31.270736

Figure Lengend Snippet: (A) Pancreas tissue section from COVID-19 Patient 1 stained for H&E. Inset highlights fibrotic center with residual acinar cells and islet surrounding ductules. Scale bars: 3mm, inset 200µm. (B) Pancreas tissue section from COVID-19 Patient 2 stained for H&E. Inset highlights microthrombus without adjacent hemorrhages. Scale bars: 4mm, inset 400µm. (C) Pancreas tissue section of COVID-19 Patient 3 stained for H&E. Inset highlights a large, irregularly shaped pancreatic islet surrounded by fibrotic tissue. Scale bars: 4 mm, inset 200µm. (D) Representative pancreas tissue sections from three COVID-19 patients stained for ACE2, insulin (INS) and glucagon (GCG). Scale bars: 200µm. (E) SARS-CoV-2 NP observed in intralobular ducts (d) near an islet in the pancreas of COVID-19 Patient 1. Scale bars: 10µm. (F) Representative image of multiple ducts showing SARS-CoV-2 NP positivity in the pancreas of COVID-19 Patient 1. Scale bar: 20µm. See also Table S3 and Fig. S4 .

Article Snippet: For single-stained kidney, duodenum, and pancreas tissue sections ( and Fig. S2D ) and for SARS-CoV-2 NP single stained lung and pancreas sections ( and Fig. S4 ), slides were incubated overnight at 4°C with one of four primary antibodies against ACE2 (rabbit monoclonal anti-ACE2, 1:200 dilution (Abcam); rabbit polyclonal anti-ACE2, 1:2,000 dilution (Abcam); mouse monoclonal anti-human ACE2, 1:100 dilution, (R&D Systems); goat polyclonal anti-ACE2, 1:100 dilution, (R&D Systems)) or a primary antibody against SARS-CoV-2 NP (mouse monoclonal anti-SARS-CoV-2 NP, 1:50 dilution, Invitrogen).

Techniques: Staining

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: Antibodies used for immunofluorescence staining.

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Immunofluorescence, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Deficiency of the Angiotensinase Aminopeptidase A Increases Susceptibility to Glomerular Injury

doi: 10.1681/ASN.2016111166

Figure Lengend Snippet: RAS peptidase expression does not change in APA-KO mice after chronic AngII infusion. Representative images depicting the effect of chronic infusion of AngII on expression of NEP, ACE2, and APN as examined by immunofluorescence. Glomerular parietal epithelial and tubular epithelial cell distribution was noted for all three enzymes, with faint podocyte localization. No difference in degree of staining was observed across treatment groups. Ctrl, control; wt, wild type. Scale bars, 200 μm.

Article Snippet: Primary antibodies were goat anti-APA (anti–BP-1; Abcam Laboratories, Cambridge, MA), 1:167 (mouse specimens) and 1:150 (rat and human specimens); mouse anti-NEP (anti-CD10; Vector Laboratories), 1:50; goat anti-ACE2 (Santa Cruz Biotechnology, Santa Cruz, CA), 1:50; rabbit anti-APN (anti-CD13; Origene, Rockville, MD), 1:250; and mouse anti-GLEPP-1 (gift from Roger Wiggins, University of Michigan, Ann Arbor, MI), 1:50.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

doi: 10.1038/s41467-020-19055-7

Figure Lengend Snippet: a Replication kinetics of SARS-CoV-2-Nluc (IC Nluc) on Vero E6 cells. Cells were infected with IC Nluc at MOI 1.0. At given time points, cells were harvested for luciferase signal measurement. The means and standard deviations from three independent experiments are presented. b Diagram to analyze hACE2 for IC Nluc entry. c Relative luciferase signals following infection of cells that were preincubated with anti-hDPP4 or anti-hACE2 antibodies. The luciferase signals from antibody-treated groups were normalized to those from untreated groups. The means ± standard deviations from three independent experiments are presented. One-way ANOVA was performed to analyze the statistical significance. d Immunofluorescence analysis of hACE2 expression in A549-hACE2 cells. At 24 h post-seeding, the cells were fixed and stained with anti-hACE2 polyclonal antibody. One representative image from six independent measurements is shown. e Luciferase signals from IC Nluc infected-A549 and A549-hACE2 cells. Cells were infected with indicated MOIs and luciferase signals were measured at 24 h post-infection. The means ± standard deviations from 4 to 6 independent experiments are presented. Correct for multiple comparisons using the Holm–Sidak method was performed for statistical analyses.

Article Snippet: On the next day, cells were washed three times with PBS to remove any residual FBS and followed by 1-h treatment with goat anti-human ACE2 antibody (R&D Systems) or anti-hDDP4 antibody (R&D Systems) (both antibodies were prepared in OptiMEM medium to the given concentrations).

Techniques: Infection, Luciferase, Immunofluorescence, Expressing, Staining

Journal: Nature Communications

Article Title: A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

doi: 10.1038/s41467-020-19055-7

Figure Lengend Snippet: a Three-day cytotoxicity of chloroquine on Vero E6 and A549-hACE2 cells. b Three-day cytotoxicity of remdesivir on Vero E6 and A549-hACE2 cells. c EC 50 of chloroquine against SARS-CoV-2-Nluc on Vero E6 and A549-hACE2 cells. d EC 50 of remdesivir against SARS-CoV-2-Nluc on Vero E6 and A549-hACE2 cells. a – d Experiments were performed at least twice. Each time, two independent assays were performed. The plots show one of the two experiments performed in duplicates. Each replicate is shown. The four-parameter dose–response curve was fitted using the nonlinear regression method and EC 50 s were calculated in the software Prism 8. e Summary of CC 50 , EC 50 , and selectivity index (SI). The mean ± standard deviations from four independent experiments are indicated. SI = CC 50 /EC 50 .

Article Snippet: On the next day, cells were washed three times with PBS to remove any residual FBS and followed by 1-h treatment with goat anti-human ACE2 antibody (R&D Systems) or anti-hDDP4 antibody (R&D Systems) (both antibodies were prepared in OptiMEM medium to the given concentrations).

Techniques: Software