The cell sheet was fixed in 4% paraformaldehyde overnight. It was rinsed in PBS and permeabilized with 0.1% triton X100. Nonspecific binding sites were blocked with 1% bovine serum albumin (Sigma, India). The endothelial cell sheet was incubated with primary antibody mouse monoclonal to alpha-1 sodium potassium ATPase plasma membrane marker; 1 : 25 dilution, (Abcam, UK) overnight at 4°C and subsequently with FITC conjugated rabbit antimouse IgG; 1: 100 dilution (Sigma, India) for 1 h. Each incubation step was followed by washing with PBS. The cells were counterstained with Hoechst (Sigma, India) and viewed under a fluorescence microscope (Leica DMI 6000, Germany).
Fitc conjugated rabbit anti mouse igg
Manufactured by Merck Group
Sourced in United States
FITC-conjugated rabbit anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is conjugated with the fluorescent dye fluorescein isothiocyanate (FITC). This product is commonly used as a detection tool in various immunoassays and imaging techniques.
Automatically generated - may contain errors
5 protocols using fitc conjugated rabbit anti mouse igg
1
Immunofluorescence Staining of Endothelial Cells
2
Polyclonal Antibodies for P. falciparum
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The rabbit polyclonal P. falciparum (Pf)-Hsp60 antibody was purchased from LSBio (Seattle, Washington, USA). The rabbit polyclonal anti-Pf-calpain was generated as described in a previous study [27 (link)]. Antisera against GroEL raised in mice were generated in this study. The secondary goat-anti-rabbit IgG conjugated with horseradish peroxidase (HRP) was purchased from Bio-Rad (Hercules, California, USA). Cy5-conjugated goat-anti-rabbit IgG, FITC-conjugated rabbit-anti-mouse IgG, and anti-GAPDH antibody produced in rabbits were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).
3
Quantifying mGluR2 Expression in RABV-Infected HEK293 Cells
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HEK293 cells were seeded onto 6-well plates at 1×106 cells per well. The cells were then infected with RABV ERA at an MOI of 5 at 37°C for 1 h. Then, the cells were thoroughly washed with PBS and trypsinized with 0.25% trypsin (without EDTA), and the dispersed cells were collected in a 1.5-mL tube. The dispersed cells were washed three times with FACS wash buffer (PBS containing 2% FCS) and fixed with 3% paraformaldehyde at room temperature for 15 min. The fixed cells were washed three times and stained with mGluR2 mAb A1 as the primary antibody and FITC-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, MO, USA) as the secondary antibody. Uninfected cells stained with mouse IgG2a isotype antibody served as a control. All cells were analyzed by using a FC500 flow cytometer (Beckman Coulter, Indianapolis, IN, USA). Cell surface fluorescence density was measured and analyzed by using FlowJo software (FlowJo LLC, Ashland, OR, USA).
4
Antiviral Efficacy of Ginsenoside Rb2 against CSFV
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Using the FA-based virus inhibition assay, the antiviral efficacy of Rb2 against CSFV was investigated.
CSFV Shimen strain with 0.05 MOI was separated and added into the ST cell monolayers and allowed to inoculation for 2 h at 37°C. After inoculation, cells were washed with PBS and serial dilutions of ginsenoside Rb2 was added. Concentrations of compounds were from 25 to 200 μg/mL. Uninfected cell and virus treated with DMSO were used as controls. The plates were incubated at the temperature of 37°C for 72 h, cells were fixed with icy stationary liquid (acetone:methanol = 3:1) at 4°C for 20 min. The cells were further incubated with mouse anti-CSFV E2 mAb WH303 at 37°C for 1 h, washed three times with PBS and incubated with FITC-conjugated rabbit anti-mouse IgG (Sigma, USA) at 37°C for 30 min. After three washes, immunofluorescence was observed using a fluorescence microscope.
CSFV Shimen strain with 0.05 MOI was separated and added into the ST cell monolayers and allowed to inoculation for 2 h at 37°C. After inoculation, cells were washed with PBS and serial dilutions of ginsenoside Rb2 was added. Concentrations of compounds were from 25 to 200 μg/mL. Uninfected cell and virus treated with DMSO were used as controls. The plates were incubated at the temperature of 37°C for 72 h, cells were fixed with icy stationary liquid (acetone:methanol = 3:1) at 4°C for 20 min. The cells were further incubated with mouse anti-CSFV E2 mAb WH303 at 37°C for 1 h, washed three times with PBS and incubated with FITC-conjugated rabbit anti-mouse IgG (Sigma, USA) at 37°C for 30 min. After three washes, immunofluorescence was observed using a fluorescence microscope.
5
Immunofluorescence Assay for PEDV Detection
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Vero cells were infected with PEDV NH-TA2020 strain (GenBank NO. ON155919) or HN1303 strain (GenBank NO. KR080551.1 ) for 24 h and the cell cultures on coverslips were fixed in 80% acetone at 4 °C for 30 min. The cells were then stained with a PEDV monoclonal antibody (Pulike Biological Engineering Inc., Henan) at 37 °C for 1 h. After incubation, cells were washed with PBS three times and stained with FITC-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h. The cells were examined under a fluorescence microscope after washing three times with PBS.
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