Journal: Viruses

Article Title: The Isolation and In Vitro Differentiation of Primary Fetal Baboon Tracheal Epithelial Cells for the Study of SARS-CoV-2 Host-Virus Interactions

doi: 10.3390/v15040862

Figure Lengend Snippet: ALI differentiated FBTECs are permissive to SARS-CoV-2 Infection. ( A ) Box plots showing the expression (on a log scale) of host factors that regulate SARS-CoV-2 replication in ALI day 28 cultures from n = 8 FBTEC donors by bulk RNA-Seq. Each donor is represented by an asterix (*), with the bar and box representing the median expression and interquartile range (IQR) across all donors, and with whiskers extending to 1.5× the IQR in either direction from the top or bottom quartile. ( B ) Western blot analysis of ACE2 and TMPRSS2 protein levels in ALI day 28 differentiated cultures from n = 4 FBTEC donors (1–4). Cell lysates of the human airway epithelial cell line BCi-NS1.1 over-expressing human ACE2 (BCi-ACE2) were used as a positive control for both proteins. GAPDH was used as a loading control. ( C ) Experimental design. ALI day 20 cultures of FBTEC cells were infected with multiple SARS-CoV-2 variants (Washington, Beta, Delta and Omicron) at a MOI of 0.05. ( D ) Immunofluorescent staining of SARS-CoV-2 nucleocapsid (green) and the nuclei (blue, DAPI) in cultures 72 h post-infection for each SARS-CoV-2 variant (Washington, Beta, Delta and Omicron). Scale bar = 50 µm. Representative images from n = 2 FBTEC donors (1–2) are shown.

Article Snippet: The following primary antibodies were used: ACE2 (1:1000, catalog number NBP2-67692, Novus Biologicals, Centennial, CO, USA), TMPRSS2 (1:1000 dilution, catalog number NBP238263, R&D Systems, Minneapolis, MN, USA) and GAPDH (1:5000 dilution, catalog number 2118S, Cell Signaling Technologies, Danvers, MA, USA).

Techniques: Infection, Expressing, RNA Sequencing, Western Blot, Positive Control, Control, Staining, Variant Assay

Journal: Viruses

Article Title: Histopathological and Immunological Findings in the Common Marmoset Following Exposure to Aerosolized SARS-CoV-2

doi: 10.3390/v14071580

Figure Lengend Snippet: Representative images of IHC for ACE2 receptor in marmoset tissues. Positive staining was observed in the small intestine (Bar = 250 µm) ( A ), mostly in the apical border of absorptive epithelial cells and at a low level in some epithelial cells of glands. Minimal staining in epithelial cells from the large intestine (Bar = 100 µm) ( B ). The positive staining in the trachea (Bar = 100 µm) ( C ), lung (Bar = 250 µm) ( D ), and respiratory nasal mucosa (Bar = 100 µm) ( E ) was almost non-existent. However, moderate positive staining was observed in glandular epithelium within the olfactory mucosa area of the nasal cavity (arrows and insert; Bar = 250 µm) ( F ). To the contrary, there was no staining in the epithelium lining or nerve terminations. Intense staining was observed in the kidney in the apical border of kidney tubular epithelial cells (arrows; Bar = 250 µm) ( G ) and moderate reaction in immune cells within the liver (Bar = 100 µm; arrows, normally extramedullary haematopoiesis) ( H ). No reaction was observed in the spleen (Bar = 250 µm) ( I ).

Article Snippet: Anti-ACE2 rabbit monoclonal antibody (NBP267692, Novus Biologicals, Centennial, CO, USA) and TMPRSS2 rabbit polyclonal antibody (NBP293322, Novus Biologicals) were used at dilutions of 1:250 and 1:1000, respectively, and were incubated for 30 min. Leica polymer refine detection kit (DS9800, Leica Biosystems) was used, and DAB as chromogen for visualization and sections were counterstained with Harris’ haematoxylin (DS9800, Leica Biosystems).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: ACE2 Receptor and Its Isoform Short-ACE2 Are Expressed on Human Spermatozoa

doi: 10.3390/ijms23073694

Figure Lengend Snippet: Western blot analysis of ACE2. The image illustrates ( A ) the glycosylated full-length ACE2 (120 KDa) and short-ACE2 isoforms (52 KDa) recognized with anti-ACE2-1 and ( B ) the glycosylated full-length ACE2 (120 KDa) detected by anti-ACE2-2. Antibodies were incubated on the same blot after membrane stripping and re-blotting. Blots were cut prior to hybridization. Each of the six lanes contains 20 µg of protein from semen of different donors. At least four independent experiments with different donors were performed. C+: protein from mouse testis.

Article Snippet: Since anti-ACE2-1 (Abcam, ab15348) and anti-ACE2-2 (Novus, NBP2-67692) antibodies are not recognized for use in cytofluorimetry, the primary polyclonal goat anti-ACE2-3 antibody from R&D Systems (AF933), previously validated and recommended for flow cytometry application, was used for this purpose.

Techniques: Western Blot, Incubation, Membrane, Stripping Membranes, Hybridization

Journal: International Journal of Molecular Sciences

Article Title: ACE2 Receptor and Its Isoform Short-ACE2 Are Expressed on Human Spermatozoa

doi: 10.3390/ijms23073694

Figure Lengend Snippet: Representative schema of the ACE2 and short-ACE2 sequences and anti-ACE2 antibodies. Full-length ACE2 is composed of 805 amino acids (aas), while short-ACE2 is 459 aas length, thus sharing the sequence between the aas 347–805 (including the C-terminal domain). Numbers in blue correspond to the aas present in each sequence, while numbers in grey correspond to the sequence of aas missing in the short-ACE2 isoform. Three different antibodies were used: anti-ACE2-1 (abcam, ab15348) that recognizes the sequence between the aas 750–805; anti-ACE2-2 (Novus, NBP2-67692), recognizing the aas 200–230 from the full-ACE2; and anti-ACE2-3 (AF933, R&D Systems) recognizing the aas 18–640, mainly present in the full-ACE2 isoform.

Article Snippet: Since anti-ACE2-1 (Abcam, ab15348) and anti-ACE2-2 (Novus, NBP2-67692) antibodies are not recognized for use in cytofluorimetry, the primary polyclonal goat anti-ACE2-3 antibody from R&D Systems (AF933), previously validated and recommended for flow cytometry application, was used for this purpose.

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: ACE2 Receptor and Its Isoform Short-ACE2 Are Expressed on Human Spermatozoa

doi: 10.3390/ijms23073694

Figure Lengend Snippet: 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 .

Article Snippet: Since anti-ACE2-1 (Abcam, ab15348) and anti-ACE2-2 (Novus, NBP2-67692) antibodies are not recognized for use in cytofluorimetry, the primary polyclonal goat anti-ACE2-3 antibody from R&D Systems (AF933), previously validated and recommended for flow cytometry application, was used for this purpose.

Techniques: Immunocytochemistry, Fluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: ACE2 Receptor and Its Isoform Short-ACE2 Are Expressed on Human Spermatozoa

doi: 10.3390/ijms23073694

Figure Lengend Snippet: Surface expression of ACE2 in viable motile human spermatozoa selected by the swim-up procedure as evaluated by flow cytometry. Typical histograms of fluorescence intensity (FL2-H) in spermatozoa incubated with ( A ) a non-specific serum from non-immunized goat (negative control) or ( B , C ) two representative sperm samples incubated with primary goat antibody against human ACE2 (anti-ACE2-3 polyclonal antibody, R&D Systems, AF933).

Article Snippet: Since anti-ACE2-1 (Abcam, ab15348) and anti-ACE2-2 (Novus, NBP2-67692) antibodies are not recognized for use in cytofluorimetry, the primary polyclonal goat anti-ACE2-3 antibody from R&D Systems (AF933), previously validated and recommended for flow cytometry application, was used for this purpose.

Techniques: Expressing, Flow Cytometry, Fluorescence, Incubation, Negative Control