mouse anti hace2 monoclonal antibody  (Santa Cruz Biotechnology)

Journal: International Journal of Molecular Sciences

Article Title: Ultrastructural Features, Immune Response, and Junctional Proteins in the Seminiferous Epithelium of SARS-CoV-2-Infected Mice

doi: 10.3390/ijms27020691

Figure Lengend Snippet: ( A – D ): Photomicrographs of testicular sections of animals showing double immunofluorescence for hACE2 and spike in animals from CG and IG ( A – D ). Nuclear staining with DAPI. In ( A – D ), sections of seminiferous tubules at stages VII–VIII show hACE2 immunoexpression (arrows) in both groups. In ( B – D ), in addition to hACE2 (arrows), spike immunolabeling (arrowheads) is observed throughout the seminiferous epithelium of IG. In ( C , D ), enhanced spike and hACE2 immunolabeling is observed in damaged regions of the seminiferous epithelium, which show reduced height (double headed arrow) and intraepithelial spaces due to loss of germ cells (*). ( E – G ) : Photomicrographs of testicular sections of animals showing immunofluorescence for nucleocapsid protein in animals from IG. Nuclear staining with DAPI. In ( E ), seminiferous tubules at stages VII–VIII show nucleocapsid immunolabeling (arrows) in Sertoli cells (inset 1), round spermatids (inset 2), and flagellum of elongate spermatids (inset 3). In ( F , G ), nucleocapsid immunoreaction is observed in Sertoli cells’ cytoplasm and elongate spermatids (arrows) of IG. (SC) Sertoli cell nucleus. SC nucleolus (arrowheads). ( H ): A weak angiotensin II signal is observed in CG when compared to a strong signal in IG. The β-tubulin signal is observed in both groups. A significant increase in angiotensin II optical density (OD) is observed in IG when compared to CG.

Article Snippet: All sections were incubated in 2% BSA for 30 min, and incubated at 4 °C overnight with the following primary antibodies: mouse anti-hACE2 monoclonal antibody (RRID: AB_2861379, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA, SC-73668, lot: #G1222), rabbit anti-SARS-CoV-2 spike protein S1 recombinant monoclonal antibody (RRID: AB_2866477, 1:250, Invitrogen, Carlsbad, CA, USA, MA5-36247, lot: XG3635472), rabbit anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody (1:3000; EPR24334-118; Abcam, Cambridge, UK; ab271180), rabbit anti-Ki-67 monoclonal IgG antibody (1:200; Abcam, Cambridge, UK; ab16667), and rabbit anti-IFN-γ polyclonal IgG antibody (1:300, Invitrogen, cat. 95560, lot: XH3666559); mouse anti-TNF-α monoclonal IgG [52B83] antibody (1:200, Abcam, Cambridge, UK; ab1793, lot:GR3446230), rabbit anti-iNOS recombinant polyclonal IgG [RM1017] antibody (1;1500; Abcam, Cambridge, UK; ab283655, lot:GR3436095-8), mouse anti-connexin 43 monoclonal antibody (RRID: AB_10707826, 1:200; Santa Cruz Biotechnology; sc-271837), and rabbit anti-NF-kB p65 polyclonal antibody ab31481 (1:200; Abcam, Cambridge, UK; ab31481).

Techniques: Immunofluorescence, Staining, Immunolabeling

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Development of Mice Sensitized to SARS-CoV-2 Infection (A and B) To assess hACE2 expression and surface localization, 17CL-1 cells were transduced with Ad5-hACE2 or Ad5-Empty at MOI of 100 at 37°C for 4 h. hACE2 expression was monitored by western blot assay (A) or flow cytometry (B). (C) Ad5-hACE2 transduced 17CL-1 cells were infected with SARS-CoV-2 at MOI of 0.5 at 48 h post transduction, and virus titers were determined by foci forming assay (FFA) at 24, 48, and 72 hours post infection (h.p.i.). (D) Five days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2 or Ad5-Empty in 75 μL of DMEM intranasally, lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with an anti-hACE2 antibody (brown color). hACE2 protein (brown color) was detected only in Ad-hACE2-treated mice and was predominantly localized to alveolar epithelial cells. Scale bars, 467 and 94 μm, top and bottom panels, respectively. (E and F) Ad5-hACE2- or Ad5-Empty-transduced BALB/c or C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 in 50 μL of DMEM. Weight changes in 6-to-8-week old BALB/c (E) and C57BL/6 (F) mice were monitored daily (n = 5 mice per group). To obtain virus kinetics in BALB/c (E) and C57BL/6 (F) mice, lungs were harvested and homogenized at the indicated time points, and virus was titered by plaque assay. Titers are expressed as PFU/g lung tissue (n = 3 mice per group per time point). Data are representative of two independent experiments. (G) 2 d.p.i., lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with anti-SARS-CoV-2 N protein. Scale bar, 476 μm. (H) Representative Hematoxylin-eosin (HE) staining of lungs from BALB/c and C57BL/6 mice harvested at the indicated time points p.i. Scale bars, 443 and 88 μm, top and bottom panels, respectively. Asterisk, edema. (I) Summary histology scores determined at the indicated time points (n = 4 to 5 mice per group). PMN, neutrophils. (J) Photographs of lung specimens isolated from infected mice at indicated time points are shown. Arrowheads indicate regions with vascular congestion and hemorrhage.

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Infection, Expressing, Transduction, Western Blot, Flow Cytometry, Virus, Staining, Plaque Assay, Isolation

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: The Role for IFN and STAT1 Signaling in SARS-CoV-2 Infection (A) 5 days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2, C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2. Weight changes were monitored daily (n = 5 mice per group), and virus titers in the lungs were measured at the indicated time points using FFA (n = 3–4 mice per group per time point). Titers are expressed as FFU/g tissue. (B) Sections of paraffin embedded lungs from SARS-CoV-2-infected Ad5-hACE2-transduced, wild-type and genetically modified C57BL/6 mice at 4 d.p.i. were stained with hematoxylin/eosin. Scale bar, 100 μm. (C) Photographs of gross pathological lung specimens isolated from infected C57BL/6 mice at 4 d.p.i. Arrowheads indicate regions with vascular congestion and hemorrhage. (D) Ad5-hACE2-transduced C57BL/6 mice were treated with 80 μg of poly I:C in 50 μL of PBS 6 h before intranasal infection with SARS-CoV-2. Weight changes were monitored daily, and viral titers in lungs were measured at the indicated time points. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Infection, Transduction, Virus, Genetically Modified, Staining, Isolation

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Requirements for T Cells and Antibodies for SARS-CoV-2 Clearance and Protection from Subsequent Challenge Ad5-hACE2-transduced mice were infected with 1 × 10 5 PFU of SARS-CoV-2. (A) For systemic depletion of CD4 + or CD8 + T cells, mice were injected intraperitoneally (i.p.) with 0.5 mg anti-CD4 antibody (clone GK1.5) and/or 0.5 mg anti-CD8 antibody (clone 2.43) or 0.5 mg rat IgG at days −2 and 0 p.i. Virus titers in the lungs were measured at the indicated time points. Titers are expressed as FFU/g tissue (n = 4 mice per group per time point) (B–D) To identify SARS-CoV-2 T cell responses, single-cell suspensions were prepared from the BALF of transduced/infected BALB/c mice and stimulated with 2 μM structural protein peptide pools for 5–6 h in the presence of brefeldin A. Flow plots (B, 7 d.p.i), and summary of frequencies and cell numbers of SARS-CoV-2-N pool specific CD4 + T cells (C) and S1 pool specific CD8 + T cells (D) (determined by IFN-γ intracellular staining) are shown (n = 3 to 4 mice per time point). (E) PRNT 50 titers in the sera of transduced/infected C57BL/6 mice at indicated time points p.i. are shown. (F) BALB/c and C57BL/6 mice were immunized with 1 × 10 5 infectious units (IU) of VRP-S intranasally in 50 μL of PBS. Mice were transduced and infected with 1 × 10 5 PFU of SARS-CoV-2 3 weeks after vaccination. Virus titers in the lungs were measured at the indicated time points (n = 4 mice per group per time point). (G) For adoptive transfer of serum, BALB/c mice were immunized with 1 × 10 5 IU of VRP in the footpad in 50 μL of PBS and boosted with the same dose 3 weeks later. Sera were obtained 1–2 weeks after VRP booster. Then, 150 μL of serum was transferred into transduced mice intravenously (i.v.) 1 day before SARS-CoV-2 infection (n = 3 mice per group per time point). ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Infection, Injection, Virus, Staining, Adoptive Transfer Assay

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Convalescent Plasma from COVID-19 Patients and Remdesivir Protect Mice from SARS-CoV-2 Infection (A and B) For plasma adoptive transfer, Ad5-hACE2-transduced mice were injected with 150 μL of plasma i.v. from a healthy donor or COVID-19, MERS, or SARS convalescent patients, at −1 d.p.i. Weight and virus titers in lung tissues were monitored (A) and expressed as FFU/g tissue (n = 4 mice per group per time point). Sections of paraffin embedded lungs from plasma adoptive transferred and infected mice were stained with HE at day 4 p.i. (B). Scale bar, 100 μm. (C and D) For remdesivir treatment, Ad5-hACE2-transduced mice were treated with remdesivir (25 mg/kg, bid s.c.) or vehicle at −1 d.p.i. Weight loss of infected mice and virus titers in the lungs were monitored (C), and hematoxylin/eosin staining of sections of paraffin-embedded lungs is shown at 4 d.p.i. (D) (n = 4 mice per group per time point). Data are representative of two independent experiments. Scale bar, 100 μm. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values of ≤ 0.0001.

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Clinical Proteomics, Infection, Adoptive Transfer Assay, Injection, Virus, Staining

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet:

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Virus, Blocking Assay, Plasmid Preparation, Software