The radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) was used to isolate the total protein. Following quantification with bicinchoninic acid kit (Thermo Fisher Scientific), 20 μg samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on the polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% fat-free milk for 1 h and then incubated with primary antibodies (E-cadherin [ab40772, 1:8000; Abcam, Cambridge, UK], intercellular adhesion molecule-1 [ICAM-1] [ab109361, 1:1000; Abcam], vitronectin [ab45139, 1:2000; Abcam], proliferating cell nuclear antigen [PCNA] [ab18197, 1:5000; Abcam], matrix metalloproteinase 9 [MMP9] [ab76003, 1:5000; Abcam], AQP4 [ab81355, 1:300; Abcam], and GAPDH [1:5000; Abcam]) overnight, followed by interacting with secondary antibodies (ab6721, 1:2000; Abcam) for 2 h. Then blots were exposed to enhanced chemiluminescence (Solarbio) and detected via ImageJ v1.8, with GAPDH as a normalized control.
Ab109361
Manufactured by Abcam
Sourced in United States
Ab109361 is an anti-SARS-CoV-2 nucleocapsid protein antibody. It is a rabbit monoclonal antibody that recognizes the nucleocapsid protein of SARS-CoV-2, the virus that causes COVID-19.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (3 mentions)
Ab76003, by Abcam (2 mentions)
Ab134047, by Abcam (2 mentions)
Ab9669, by Abcam (2 mentions)
Ab137867, by Abcam (2 mentions)
Ab178945, by Abcam (2 mentions)
Gapdh, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Bicinchoninic acid kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab45139, by Abcam (1 mentions)
Ab18197, by Abcam (1 mentions)
Gapdh, by Solarbio (1 mentions)
F2761, by Thermo Fisher Scientific (1 mentions)
Fluoroshield, by Merck Group (1 mentions)
Ab6800, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
9 protocols using ab109361
1
Western Blot Analysis of Cell Adhesion and Proliferation Markers
2
Immunofluorescence Analysis of SARS-CoV-2 Proteins
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Ten-micrometer-thick cryosections were double-stained using a monoclonal mouse anti-SARS-CoV-2 antibody (1:1000 dilution, #MBS569903; MyBioSource, San Diego, CA, USA) and one of the following antibodies: polyclonal rabbit anti-pan cytokeratin antibody (1:100 dilution, #ab9377; Abcam), monoclonal rabbit anti-CD14 antibody (1:100 dilution, #ab18332; Abcam), polyclonal rabbit anti-ACE2 (1:100 dilution, recognizing both short and long forms of ACE2; #PK-AB718-3217, PromoCell, Heidelberg, Germany), monoclonal rabbit anti-ICAM-1 (1:100 dilution, #ab109361, Abcam). Mouse monoclonal and rabbit monoclonal or polyclonal isotype antibodies (#ab18469, #ab172730 and #ab15348; Abcam) functioned as negative controls. In the secondary step, FITC-conjugated polyclonal goat anti-mouse antibodies (1:200 dilution #F2761, ThermoFisher Scientific) were combined with Texas Red-conjugated polyclonal donkey anti-rabbit antibodies (1:100 dilution, #ab6800, Abcam). In the tertiary step, FITC-conjugated polyclonal donkey anti-goat antibodies were added (1:200 dilution, #A16006, ThermoFisher Scientific). DAPI (#D9542, Sigma-Aldrich) was used to counterstain cell nuclei. Slides were mounted with Fluoroshield™ (#F6182, Sigma-Aldrich) and analyzed using a Leica (TCS SPE) confocal microscope and Leica Las X v3.7.2.22383 software.
3
Immunofluorescence Analysis of Cell Adhesion Molecules
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The deparaffinized sections were incubated with Tris–EDTA buffer for 20 min and 3% H2O2 for 10 min. The sections were exposed to 2% BSA for 30 min at 37°C. Then, they were incubated with fluorescein-labeled primary antibodies against ICAM1 (1:200; ab109361; Abcam, United States), KLF2 (1:200; ab244507; Abcam, United States), and VCAM1 (1:200; ab134047; Abcam, United States) for 4 h at room temperature. The results were investigated under an immunofluorescence microscope (Olympus, Japan).
4
Protein Expression and Western Blot Analysis
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Total protein was extracted using RIPA lysis buffer containing PMSF on ice for 30 min. The lysate was centrifuged at 4°C, 8000g for 10 min, then with the supernatant collected. Total protein concentration was tested with a BCA kit. Protein (50 μg) was dissolved in 2× SDS loading buffer, boiled at 100°C for 5 min, and subjected to SDS‐polyacrylamide gel electrophoresis. The protein was transferred to a PVDF membrane by wet transfer and 5% skimmed milk was used for blocking (room temperature, 1 h). Then the PVDF membrane was incubated with diluted primary antibodies against MMP‐9 (ab137867, 1:1000, Abcam), iNOS (ab178945, 1:1000, Abcam), ICAM‐1 (ab109361, 1:1000, Abcam), MCP‐1 (ab9669, 1:1000, Abcam), and GAPDH (ab8245, 1:2000, Abcam) overnight at 4°C, washed, and incubated with horseradish peroxidase‐labeled secondary antibody goat anti‐rabbit IgG (1:5000, Beijing ComWin Biotech Co., Ltd.) for 2 h at room temperature. Electrochemiluminescence was used for color development and finally analyzed on a gel imager. The gray level of the bands was analyzed using ImageJ software. The experiment was repeated three times.
5
Quantitative Protein Analysis via Western Blotting
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Total protein was extracted using RIPA lysis buffer containing PMSF on ice for 30 min. The lysate was centrifuged at 4°C, 8000g for 10 min, and then the supernatant was collected. Total protein concentration was tested with a BCA kit. Protein (50 μg) was dissolved in 2 × SDS loading buffer, boiled at 100°C for 5 min, and subjected to SDS‐polyacrylamide gel electrophoresis. The protein was transferred to a PVDF membrane by wet transfer and 5% skimmed milk was used for blocking (room temperature, 1 h). Then the PVDF membrane was incubated with diluted primary antibodies against SAMD1 (PA5‐65308, 1:1000; Thermo Fisher Scientific), MMP‐9 (ab137867, 1:1000; Abcam), iNOS (ab178945, 1:1000; Abcam), ICAM‐1 (ab109361, 1:1000; Abcam), MCP‐1 (ab9669, 1:1000; Abcam) and GAPDH (ab8245, 1:2000; Abcam) overnight at 4°C, washed, and incubated with horseradish peroxidase‐labeled secondary antibody goat antirabbit IgG (1:5000; Beijing ComWin Biotech Co., Ltd) for 2 h at room temperature. Electrochemiluminescence (ECL) was used for color development and finally analyzed on a gel imager. The gray level of the bands was analyzed using Image J software. The experiment was repeated three times.
6
Glucose-Induced Endothelial Cell Migration
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ECs were pre-treated with high (25 mM) or low (5 mM) glucose concentrations for 6 days. In some cases, anti-ICAM-1/LFA-1 neutralizing antibody (ab109361, ab52895; Abcam, Cambridge, MA, United States) was added to the culture medium and confluent monolayer cells were scraped off using a 200-μL pipette tip. For the in vitro assay, we gently removed the debris, cleaned the scratch border and replaced the volume with growth medium (Becker et al., 1991 (link)). To determine the number of ECs that had migrated into the scraped area, photographs were taken at various times and analyzed using NIS-Elements D image analysis software (Nikon, Tokyo, Japan).
7
Quantifying Apoptosis Markers in VSMCs
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VSMCs from each group were collected and lysed on ice using lysis buffer (Solarbio, Beijing, China). The mixture was centrifuged at 12,000 rpm at 4°C for 15 minutes. After discarding the supernatant, the protein concentration was determined using a BCA kit (Merck, Kenilworth, NJ, USA). Equal amounts of protein were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane, which was then blocked with 5% skimmed milk at 25°C. The membrane was then incubated at 4°C overnight with the following primary antibodies, which were all purchased from Abcam: anti-Bax (ab3191, 1 : 2000), anti-Bcl-2 (ab196495, 1 : 1000), anti-vascular cell adhesion molecule 1 (VCAM-1; ab174279, 1 : 2000), anti- intercellular cell adhesion molecule-1 (ICAM-1; ab109361, 1 : 2000), anti-E-selectin (ab137732, 1 : 2000), anti-GBP-1 (ab22604, 1 : 2000), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, 1 : 5000). The next day, the membrane was rinsed and incubated with the secondary antibody at 37°C for 30 minutes. Enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) was used to visualize the protein bands and GAPDH was used to normalize the protein levels.
8
Adenoviral Protein Expression Profiling
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At 48 h post adenovirus infection, whole-cell lysates were prepared utilizing RIPA buffer (Sigma-Aldrich, St. Louis, USA). Protein concentrations were determined via a BCA Protein Assay kit (Pierce). Equal amounts of total proteins (20 µg) were loaded onto gels, separated through SDS-PAGE, and transferred to polyvinylidene difluoride membranes (PVDF) (Merck KGaA, Darmstadt, Germany). Signals were detected using the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore, MA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the loading control. The primary antibodies employed included: anti-p-JAK2 (1:1000, #3771, CST), anti-p-STAT1 (1:1000, #9167, CST), anti-p-STAT3 (1:1000, #9145, CST), anti-PDGFRB (1:1000, #3169, CST), anti-α-SMA (1:1000, #19,245, CST), anti-SM22α (1:1000, ab14106, Abcam), anti-MMP1 (1:1000, ab137332, Abcam), anti-MMP9 (1:1000, ab76003, Abcam), anti-VCAM1 (1:1000, ab134047, Abcam), anti-ICAM1 (1:1000, ab109361, Abcam), anti-GAPDH (1:2500, ab9485, Abcam), anti-p-Src (1:1000, #6943, CST), anti-p-PLCγ (1:1000, #14,008, CST), anti-p-Erk1/2 (1:1000, #4370, CST).
9
Immunofluorescence Staining for NF-κB and ICAM1
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HCECs were fixed with 4% formaldehyde for 20 min at room temperature (RT), permeated with 0.5% Triton X-100 for 10 min and blocked with 5% bovine serum albumin (BSA) for 1 h. Afterwards, the cell slides were incubated with primary antibody against NF-κB p65 (1:100, ab32536, Abcam), ICAM1 (1:100, ab109361, Abcam) overnight at 4 °C. Next day, secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit (Thermo Fisher Scientific, USA) incubated for 1 h at RT. After washing with PBS, the cell slides were incubated with DAPI for nuclear staining and mounted with anti-fade mounting medium. The slides were observed using a laser scanning confocal microscopy (Leica, TCS SP8 SR, Germany).
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