We performed western blot analyses, as described previously [29 (link)]. In this context, we briefly lysed the cells in lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM EDTA, 50 mM NaF, 50 mM β-glycerophosphate, 1 mM Na3VO4, and 25 μg/mL each of antipain, leupeptin, and pepstatin) for 30 min on ice. After centrifugation at 20,400 g for 10 min at 4°C, we collected the lysates, separated them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred them onto polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany), and analyzed them by Western blotting. We measured protein levels using the following primary antibodies: anti-ACE2 antibody (ab273433, 1:1000; Abcam, Cambridge, UK) and anti-β-tubulin antibody (#5346, 1:1000; Cell Signaling Technology, Danvers, MA, USA).
Ab273433
Manufactured by Abcam
Sourced in United Kingdom
Ab273433 is a lab equipment product. It is a sterile, single-use pipette tip compatible with most common pipette models.
Automatically generated - may contain errors
Lab products found in correlation
Anti β tubulin antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab228549, by Abcam (1 mentions)
Ab271180, by Abcam (1 mentions)
Lsm 880 meta confocal microscope, by Zeiss (1 mentions)
Ab108252, by Abcam (1 mentions)
Dmirb fluorescence microscope, by Leica (1 mentions)
Cy3 conjugated goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab28364, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Nacalai Tesque (1 mentions)
Slowfade, by Thermo Fisher Scientific (1 mentions)
Gtx135357, by GeneTex (1 mentions)
Ab273434, by Abcam (1 mentions)
Anti c5b 9, by Agilent Technologies (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Dab elite kit, by Agilent Technologies (1 mentions)
Anti ace2, by Sino Biological (1 mentions)
7 protocols using ab273433
1
Western Blot Analysis of ACE2 Protein
2
SARS-CoV-2 Spike Protein Immunohistochemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For routine histology, the tissue paraffin sections were stained with H&E. For IHC, lung tissues sections were incubated with the primary antibodies to Spike (ab273433, Abcam, 1:1000), Spike (A20022, ABclonal, 1:1000).[78 ] The quantification of ACE2 protein expression was obtained by measuring the H score. The H‐score was calculated as: the formula 3 × the percentage of strongly staining cells + 2 × the percentage of moderately staining cells + the percentage of weakly staining cells, yielding an H‐score range of 0 to 300. For IF, after re‐hydration and antigen retrieval, tissue sections were blocked by 3% BSA for 1 h, and then stained with specific antibodies, followed by incubation with secondary antibodies. Nuclei were stained with DAPI.
3
Investigating SARS-CoV-2 Antiviral Activity and Cell Fusion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the analysis of the antiviral activity of compounds, cells were preincubated with compounds, infected, and incubated for 48 h prior to immunofluorescence staining. For the cell-cell fusion assay, 293T/hACE2 cells were incubated with 293T cells transfected with the SARS-CoV-2 spike protein in the presence or absence of ATRA. The cocultured cells were incubated for 24 h, and the state of cell fusion was observed by microscopy and immunofluorescence staining.
Cells were washed three times in PBS and fixed with 4% paraformaldehyde (PFA) for 2 h at room temperature. Then, the cells were permeabilized with 0.1% Triton X-100 for 30 min, washed with PBS, blocked with 2% bovine serum albumin (BSA) for 1 h, and stained with anti-SARS-CoV-2 nucleocapsid (Abcam catalog no. ab271180), anti-SARS-CoV-2 spike (Abcam catalog no. ab273433), and anti-hACE2 (Abcam catalog no. ab108252) antibodies for 2 h at room temperature, according to different experimental settings. The cells were washed with PBS and then stained with secondary antibodies for 1 h at room temperature. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (Abcam catalog no. ab228549). Images were examined by a Zeiss LSM 880 Meta confocal microscope.
Cells were washed three times in PBS and fixed with 4% paraformaldehyde (PFA) for 2 h at room temperature. Then, the cells were permeabilized with 0.1% Triton X-100 for 30 min, washed with PBS, blocked with 2% bovine serum albumin (BSA) for 1 h, and stained with anti-SARS-CoV-2 nucleocapsid (Abcam catalog no. ab271180), anti-SARS-CoV-2 spike (Abcam catalog no. ab273433), and anti-hACE2 (Abcam catalog no. ab108252) antibodies for 2 h at room temperature, according to different experimental settings. The cells were washed with PBS and then stained with secondary antibodies for 1 h at room temperature. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (Abcam catalog no. ab228549). Images were examined by a Zeiss LSM 880 Meta confocal microscope.
4
SARS-CoV-2 Spike Protein Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vero cells were infected with various rMVs at MOI of 0.1. At 24–36 h post-infection, cells were fixed with 4% paraformaldehyde, blocked with 2% goat serum overnight, and then treated with or without 0.1% saponin A (Sigma). Fixed cells were probed with a mouse monoclonal anti-SARS-CoV S antibody (ab273433, Abcam, 1:300 dilution) and followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (A-11008, Thermo Fisher). Staining with anti-MV-N followed by Cy3-conjugated goat anti-rabbit (A10520, Jackson ImmunoResearch, 1:1000 dilution) was used to detect MV in the same infected cells. Nuclei were stained with DAPI. Images were collected using an inverted Leica DM IRB fluorescence microscope with a ×20 objective.
5
SARS-CoV-2 Infection Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were nucleofected with Ctrl, TIP1 or TIP2 RNA and plated with 80% confluency in a 96-well black/clear bottom plate. At 24 hours post nucleofection, cells were infected with SARS-CoV-2 (WA-1 strain) at MOI = 20, cells were washed three times with 100 μL sterile PBS buffer after 1 hour of infection, at 2 hours post infection, cells were fixed with 4% paraformaldehyde for 15 minutes, followed by washing wells twice with 100 μL of PBS buffer. Cells were permeabilized by adding 0.5% Triton X-100 in PBS for 5 minutes, followed by 1x washing with PBS, blocking with 3% BSA in PBS buffer for 30 minutes at room temperature. Wells were washed with PBS, followed by addition of 30 μL of primary antibody (mouse anti-Spike antibody, cat# ab273433, Abcam, RRID:AB 2891068) for one hour, followed by 100 μL of secondary antibody (anti-mouse Alexa flour 488) in 3% BSA in PBS buffer for 30 minutes, wells were washed twice with PBS and 100 μL of 1:2000 Hoechst stain was added for 10 minutes, followed by two washes in PBS. High-throughput microscopy was performed on an ImageXpress-Confocal Microscope, and images were analyzed using MetaXpress software.
6
Immunocytochemistry of SARS-CoV-2 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunocytochemistry was performed as previously described [20 (link)]. Briefly, the cells were fixed, permeabilized, blocked, and incubated with primary antibodies against SARS-CoV-2 nucleocapsid (#GTX135357, 1:100; GeneTex, Irvine, CA, USA), SARS spike glycoprotein (1A9, #ab273433, 1:100; Abcam, Cambridge, UK), CD31 (#ab28364, 1:25; Abcam) [29 (link)], CLDN5 (4C3C2, #35-2500, 1:25; Thermo Fisher Scientific) [29 (link)], cleaved caspase-3 (5A1E, #9664S, 1:500; Cell Signaling Technology), phospho-GYS1 (1D1, # CSB-RA010078A641PHHU, 1:100; Cusabio, Wuhan, China), and β-catenin (15B8, #37447S, 1:3000; Cell Signaling Technology, Danvers, MA, USA) at 4 °C. The cells were then incubated with Alexa 488-conjugated (1:200; Thermo Fisher Scientific) or Alexa 594-conjugated (1:200; Thermo Fisher Scientific) secondary antibodies for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole DAPI (Nacalai Tesque Inc.). The cells were mounted in SlowFade (Thermo Fisher Scientific) and examined under a confocal laser-scanning microscope (Nikon A1; Nikon, Tokyo, Japan).
7
Immunohistochemical Detection of SARS-CoV-2 Antigens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protocol for immunohistochemistry has been reported in our previous work45 (link). Briefly, antigen retrieval was performed by microwaving these sections in citrate buffer (10 mM, pH 6.0). The sections were then incubated in 3% BSA plus 0.1% H2O2 for 1 h at RT to block nonspecific binding. The sections were then incubated overnight at 4 °C with primary anti-SARS-CoV-2 NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-SARS-CoV-2 NP antibodies (ab273434, 1:500, mouse monoclonal 6H3, Abcam), anti-SARS spike glycoprotein (S) antibodies (ab273433, 1:500, mouse monoclonal 1A9, Abcam), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological), anti-CD8 (Clone ID:4B11, 1:100, mouse IgG2b; BIO-RAD), anti-CD68 (Clone ID:KP1, 1:100, mouse IgG1; BIO-RAD), anti-CD56 (Clone ID:123C3, 1:100, mouse IgG1; BIO-RAD), anti-C5b-9 (clone ID: aE11, 1:100, mouse IgG; Dako cytomation). Sections were further incubated with the Goat anti-Rabbit IgG (H + L) secondary antibody, HRP (#31460, Invitrogen) or Goat anti-Mouse secondary antibody, HRP (PA1-74421, ThermoFisher) for 1 h at RT, respectively. Peroxidase activity was visualized with the DAB Elite kit (K3465, DAKO), and the brown coloration of tissues represented positive staining as viewed by a light microscope (Zeiss Axioplan 2, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!