A549 cells were infected at an empirically determined MOI (by titration) that gave ~10% levels of infection for 6 hr, after which cells were fixed, permeablised and stained for a range of viral proteins. M2 was detected by ab5416 (Abcam, Cambridge, UK), NS1 by an in-house (rabbit) antiserum, NA by an in house (mouse) antiserum, NP by ab20343 (Abcam, Cambridge, UK) and PB2 by an in house rabbit antiserum. Secondary antibodies were species specific Alexafluor-488 or Alexafluor-546 (A21202, A11005, A21207 and A21206, Life Technologies, Loughborough, UK).
Ab20343
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab20343 is a laboratory equipment product manufactured by Abcam. It is a general-purpose tool used in research and scientific applications. The core function of this product is to facilitate specific tasks or experiments conducted in a laboratory setting. No further details about its intended use or capabilities are provided.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Ab6046, by Abcam (3 mentions)
A21206, by Thermo Fisher Scientific (2 mentions)
A21202, by Thermo Fisher Scientific (2 mentions)
Atto647n conjugated anti mouse antibody, by Merck Group (2 mentions)
Alexafluor488 af488 conjugated anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Ab205719, by Abcam (2 mentions)
Ab20351, by Abcam (2 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
A11005, by Thermo Fisher Scientific (1 mentions)
Ab5416, by Abcam (1 mentions)
Zg003, by Thermo Fisher Scientific (1 mentions)
Ab219800, by Abcam (1 mentions)
Ab3280, by Abcam (1 mentions)
A32965, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
Anti sars cov 2 nucleocapsid antibody, by Sino Biological (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
19 protocols using ab20343
1
Quantifying Viral Protein Expression
2
Immunofluorescence Staining of Cultured Cells
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Cells in 2D or 3D cultures were treated with 4% (w/v) paraformaldehyde and then permeabilized with 0.5% Triton X-100. The first blotting antibodies (mouse antibody against mucin 5A, Abcam; mouse antibody against p63, Abcam; mouse antibody against cytokeratin 14, Abcam; and mouse antibody against influenza A virus nucleoprotein, Abcam ab20343) were incubated according to the manufacturer's procedures and previous studies [23 (link), 25 (link), 27 (link)]. 0.5 μg/ml DAPI (D3571) was used to stain the nuclei. All images were captured by the Leica DM4000B fluorescence microscope.
3
Detecting Protein Expression in Lung Tissue
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For detection of protein expression in lung tissue, samples were lysed in a 1% SDS buffer (1% SDS, 50mM triethanolamine pH 7.4, 150mM NaCl) containing a cocktail of phosphatase protease inhibitors (Sigma, 4906845001) and protease inhibitors (Thermo Scientific, A32965). The lysates were centrifuged at 15,000 rpm for 10 min and soluble protein supernatents were used for Western blot analysis. Equal amounts of protein (30ug) were separated by SDS-PAGE and transferred onto membranes. Membranes were blocked with 10% non-fat milk in Phosphate-buffered saline with 0.1% Tween-20 (PBST) and probed with antibodies against GSDMD (abcam, ab219800) and actin (abcam, ab3280). For Western blotting of THP-1 cells, samples were lysed in 1% SDS containing protease inhibitors prior to SDS-PAGE separation as described above. Membranes were probed with antibodies against influenza virus nucleoprotein (abcam, ab20343), GSDMD (abcam, ab210070), and GAPDH (Thermo Scientific, ZG003).
4
Flow Cytometric Analysis of Influenza Virus NP
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A549 (6 × 105) or Calu-3 (24 × 105) cells seeded in six-well plates were mock infected or infected with the indicated viruses. Cells were harvested with trypsin, followed by fixation (4% paraformaldehyde) and permeabilization (0.1% Triton X). Cells were then stained with a primary mouse monoclonal anti-influenza virus NP antibody (Abcam; ab20343) and a secondary anti-mouse DyLight633 (red) antibody (Thermo Fisher; 35512). The stained cells were subjected to FACS analysis (Attune NxT Flow Cytometer). Data were analyzed and processed using the FlowJo software.
5
Quantitative Imaging of SARS-CoV-2 and IAV in Cells
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The rehydrated cells were stained with anti-SARS-CoV-2 spike antibody CR3022 from MassBiologics [12 (link)] or with anti-SARS-CoV-2 nucleocapsid antibody (Sinobiological Cat #40143-MM05, Wayne, PA, USA) conjugated with AlexaFluor-488 at 1:200 for 1 h at room temperature. Once the staining was complete, the cells were washed with PBS, and then, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/mL, Abcam, Boston, MA, USA) for 15 min to visualize cell nuclei. Fluorescence from the cells was detected using the ImageXpress® Micro-XL (IXM) Automated Imaging System. The IXM software was used to count the total numbers of AlexaFluor-488-positive cells and DAPI-positive cells. The same protocol was applied for IAV staining, using an anti-IAV nucleocapsid antibody (AbCam Cat #ab20343, Boston, MA, USA) and goat anti-mouse antibody conjugated with AlexaFluor-488 (Invitrogen Cat #A11029, Waltham, MA, USA). The images were processed by MetaXpress Software.
6
Influenza Virus Binding Assays in Tissue Sections
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Virus binding assays with a low pathogenic AIV (LPAIV) H5N2 (A/H5N2/chicken/Pennsylvania/7659/1985) and human pandemic H1N1 virus (A/H1N1/Virginia/2009) were performed as previously described14 (link). Virus binding assays were performed following strict biosafety level-2 (BSL-2) practices. Briefly deparaffinised tissue sections were incubated with a 250 μl of LPAI H5N2 virus (106 TCID50/ml) or human H1N1 virus (106 TCID50/ml) for 2 h at RT. Tissues incubated with PBS served as mock treated controls. Sections were washed with TBS before blocking with inactivated goat serum for 30 min and immunostained with primary mouse monoclonal antibody to influenza hemagglutinin H5 (ab82455, Abcam) or influenza nucleoprotein (ab20343, Abcam). Following 40 min of incubation with the primary antibody, sections were washed and incubated with a secondary goat anti mouse IgG-Cy5 antibody (ab6563, Abcam). After 40 min of incubation with secondary antibody at RT, sections were washed with TBS and mounted in ProLongGold antifade reagent with DAPI. Following 24 h of curing, the sections were imaged using Olympus FluoView™ FV1000 confocal microscope.
7
Immunological Analysis of Influenza Antibodies
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The primary antibodies used were as follows: SH487 sheep serum anti-HA of Brisbane/10 and SH510 sheep serum anti-HA of A/California/07/2009 (both used at 1: 1000 for western blot and 1: 100 for immunofluorescence) were a kind gift from Mr Robert Newman (NIBSC); PE78 mouse monoclonal anti-NA of A/California/04/2009 antibody (used at 1: 100 for immunofluorescence) was a kind gift from Dr Richard Webby (St Jude Children's Research Hospital, Memphis, USA); R10/8 rabbit serum anti-X-31 (used at 1: 100 for immunofluorescence) was obtained from the WHO CC NIMR; mouse monoclonal anti-nucleoprotein (NP) antibody (ab20343, AbCam) was diluted at 1: 1000 for immunostaining virus plaques. Secondary antibodies used were as follows: goat anti-rabbit Alexa Fluor 488, donkey anti-sheep Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 (all from Invitrogen) were used at a dilution of 1: 1000. Goat anti-mouse horse radish peroxidase conjugate antibody (Bio-Rad, 172-1011) was used at a dilution of 1: 1000 for immunostaining plaques. Rabbit anti-sheep Dylight 800 antibody was used at 1: 5000 for western blotting. Ferret sera for HA inhibition (HI) assays, ferret anti- Brisbane/10 and ferret anti-Lviv, both receptor-destroying-enzyme treated, were obtained from the WHO CC NIMR.
8
Antibody Detection of Influenza Proteins
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Polyclonal antibodies were used to detect TRIM22 (Sigma-Aldrich; HPA003575), Mx1 (Santa Cruz; sc-50509), GBP1 (Proteintech; 15303-1-AP), IFIH1 (Proteintech; 21775-1-AP), TLR3 (Proteintech; 17766-1-AP), and actin (Sigma-Aldrich; A5060). IAV hybridoma antisera were used to detect NP, M1, and NS1, as previously described (Turnbull et al., 2016 (link)). Monoclonal antibodies were used to detect UBA7 (AbCam; ab133499), Actin (DSHB; 224-236-1), IFITM1 (Proteinech; 60074-1g), and IAV NP (AbCam; ab20343). Secondary antibodies were Alexa 488 and 555 donkey anti-mouse and -rabbit (Invitrogen; A21202, A21206, and A31572), DyLight 680- or 800-conjugated anti-rabbit (Thermo Fisher Scientific; 35568 and SA5-35571), and peroxidase conjugated anti-mouse (Sigma-Aldrich; A4416).
9
Immunofluorescence Assay for Viral Proteins
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As described previously (Liao et al., 2019 (link)), cells transiently expressing the target proteins were fixed with 1.875% formaldehyde, permeabilized with 0.5% NP-40, and then incubated with anti-NS1 (sc-130568, Santa Cruz Biotechnology) antibody or anti-NP antibody (ab20343, Abcam) for 1 h. Cells were further incubated with corresponding secondary antibodies conjugated with Alexa Fluor 488 (Invitrogen) for an additional 1 h at room temperature. Washing (PBS containing 1% FBS) was conducted between each step for a total of six washes. Subsequently, cells were treated with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) at a final concentration of 1 μg/ml for nucleic acid counterstaining. Images were acquired by confocal microscopy (FV1000, Olympus, Tokyo, Japan) with Olympus FV10-ASW 1.3 viewer software.
10
Antibody Reagents for Influenza Studies
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Sheep polyclonal anti-TGN46 (1:200; IFM; BioRad); mouse monoclonal anti-β-actin (A5316; 1:1,000; Sigma-Aldrich; IB); rabbit polyclonal anti-PI4KIIβ (1:1,000; IB) and -PI4KIIα (1:1,000; IB) were generous gifts from Pr. Pietro De Camilli (Yale School of Medicine, New Haven, CT); rabbit polyclonal anti-β-tubulin (ab6046; 1:1,000; Abcam; IB); rabbit polyclonal anti-pallidin (10891-1-AP; 1:1,000; Proteintech; IB); goat polyclonal anti-VPS35 (ab1099; 1:1,000; Abcam; IB); mouse monoclonal anti-SNX1 (#611482; 1:1,000; BD Bioscience; IB); mouse monoclonal anti-SNX2 (#611308; 1:1,000; BD Bioscience; IB); rabbit polyclonal anti-α-calnexin (ADI-SPA-860D; 1:1,000; Enzo Life Sciences; IB); rabbit polyclonal anit-RAB11A (#15903-1-AP; 1:200; IFM; Proteintech); mouse monoclonal anti-NP (ab20343; 1:1,000; Abcam, IF); rabbit polyclonal anti-NP (1:1,000), -PB1 (v19/6; 1:500), -PB2 (2N580; 1:500), -PA (1:500), and -NS1 (v29; 1:500) for IB were kindly provided by Pr. Paul Digard (Roslin Institute, Edinburgh, UK); mouse monoclonal anti-M2 (ab5416, clone 14C2; 1:500; Abcam; IB); mouse monoclonal anti-actin (#A5441; 1:1,000; Sigma-Aldrich; IB); goat anti-M1 (ab20910; 1:500; Abcam; IB); goat polyclonal anti-GST (#27-4577-01; 1:2,000; GE healthcare, IB); and rabbit polyclonal anti-Cap1 (1 μg/ml, prepared as described in Hamaoui et al. [2020] (link)).
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