Bacteria were treated ± antibiotics as described above for 2 h prior to culture in 90% NHS for 30 min at 37°C. Deposition of serum complement component C5b-9 on K. pneumoniae was measured by flow cytometry as described previously (17 (link)). Bacteria were pelleted by centrifugation at 1,000 × g for 10 min at 4°C, washed with DPBS, and incubated in 1 mL blocking buffer (5% normal goat serum in DPBS) for 1 h. Bacteria were pelleted by centrifugation and resuspended in DPBS. A 100-μL aliquot of resuspended cells was dispensed into new 1.5-mL tubes and labeled with antibodies coupled to fluorescein isothiocyanate (FITC). To detect surface-bound C5b-9, bacteria were incubated with anti-C5b-9 plus C5b-8 antibody (Abcam) in DPBS followed by FITC-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). An FITC-conjugated mouse IgG2a kappa was used as an isotype control antibody (Ab) (eBioscience, ThermoFisher Scientific, Waltham, MA). Antibody labeling was done on ice for 30 min followed by the addition of wash buffer (2% goat serum in DPBS) and centrifugation at 1000 × g for 10 min at 4°C. Cell pellets were resuspended in 200 μL of wash buffer and analyzed quantitatively by flow cytometry by using a BD FACSCelesta cell analyzer (BD Bioscience, San Jose, CA).
Fitc conjugated anti mouse igg antibody
Manufactured by Jackson ImmunoResearch
Sourced in Panama, United States
FITC-conjugated anti-mouse IgG antibody is a laboratory reagent used for the detection and visualization of mouse immunoglobulin G (IgG) in various immunoassays and research applications. The antibody is conjugated to the fluorescent dye FITC (Fluorescein isothiocyanate), which allows for the specific labeling and detection of mouse IgG molecules.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Tcs sp5 confocal system, by Leica (2 mentions)
Facscelesta cell analyzer, by BD (1 mentions)
Fitc conjugated anti mouse c3 antibody, by MP Biomedicals (1 mentions)
B5002, by Merck Group (1 mentions)
Rnase a, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Anti brdu antibody, by BD (1 mentions)
Isotype control igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using fitc conjugated anti mouse igg antibody
1
Quantifying Complement Deposition on Klebsiella
2
Quantifying TLR2 Clustering in RAW264.7 Cells
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RAW264.7 cells were treated with or without 10 μg/mL LTPa for 1 h at 37°C. The cells were then incubated (30 min, on ice) with anti-mouse monoclonal antibody TLR2 or isotype control (IgG) (eBioscience) before washing with PBS. Then, the cells were stained with a FITC–conjugated anti-mouse IgG antibody for 30 min on ice (Jackson Laboratories). After washing with PBS and fixing with paraformaldehyde, the cell nuclei were stained with DAPI (1:500 dilution in PBS) (Sigma-Aldrich). The cells were then imaged with the Leica TCS SP5 confocal system (Leica Microsystems Ltd.). LAS AF software was used to process the images and quantify the fluorescence. In addition, to generate a fluorescence histogram profile, a line was drawn along the cell surface. Fluorescence intensities higher than 40 arbitrary units (isotype control staining) were considered clusters of TLR2 molecules.
3
Urinary Protein Analysis in Mouse Kidney Disease
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To determine the proteinuria level, the urine of mice with the indicated genotype was collected and tested using the urine strip at specified time points (Macherey-Nagel). The proteinuria index was as follows: 0 for 0 mg/dL, 1 for 0-30 mg/dL, 2 for 30 mg/dL, 3 for 30-100 mg/dL, 4 for 100 mg/dL, 5 for 100-500 mg/dL, and 6 for values greater than 500 mg/dL.
Mouse kidneys were excised and embedded in the optimum cutting temperature (OCT) compound after being euthanized and were serially cut into 7 μm thick sections. The frozen kidney sections were fixed with precooled acetone (-20°C) at room temperature and then stained with the FITC-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch) and the FITC-conjugated anti-mouse C3 antibody (MP Biomedical). Images were taken at 200× magnification using a fluorescence microscope. We analyzed the fluorescence intensity in each glomerulus using the ImageJ software (at least 25 glomeruli were analyzed per mice). The paraffin-embedded kidney sections were stained with periodic acid-Schiff (PAS) by NTUCM Laboratory Animal Center (Taipei, Taiwan). The 400-fold images were taken, and the percentage and total intensity of the PAS-positive area in the individual glomerulus were analyzed by ImageJ software (at least 20 glomeruli/mouse were analyzed). The PAS staining score was calculated by percentage (0 to 100) × total intensity/100000.
Mouse kidneys were excised and embedded in the optimum cutting temperature (OCT) compound after being euthanized and were serially cut into 7 μm thick sections. The frozen kidney sections were fixed with precooled acetone (-20°C) at room temperature and then stained with the FITC-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch) and the FITC-conjugated anti-mouse C3 antibody (MP Biomedical). Images were taken at 200× magnification using a fluorescence microscope. We analyzed the fluorescence intensity in each glomerulus using the ImageJ software (at least 25 glomeruli were analyzed per mice). The paraffin-embedded kidney sections were stained with periodic acid-Schiff (PAS) by NTUCM Laboratory Animal Center (Taipei, Taiwan). The 400-fold images were taken, and the percentage and total intensity of the PAS-positive area in the individual glomerulus were analyzed by ImageJ software (at least 20 glomeruli/mouse were analyzed). The PAS staining score was calculated by percentage (0 to 100) × total intensity/100000.
4
BrdU Incorporation and Cell Cycle Analysis
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Cells were cultured in the presence of BrdU (final: 20 µM; Sigma B5002) for 15 min and fixed in ice-cold 70% EtOH for >1 h. Cells were then treated with 2.5 N HCl to denature DNA for 30 min. After washing with PBS containing 1% BSA, cells were incubated with anti-BrdU antibody (1:5; BD Biosciences 347580) and stained with FITC-conjugated antimouse IgG antibody (1:50; Jackson ImmunoResearch 115-095-003) and propidium iodide (final: 5 µg/mL; Sigma P4170) in the presence of RNase A (final: 0.1 mg/mL; Sigma R5503). For PI single-staining analysis without BrdU incorporation, cells were fixed in 70% ethanol and stained with propidium iodide in the presence of RNase A.
5
TLR2 Receptor Expression Imaging
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RAW264.7 cells were treated with or without 10 μg/mL CbTP for 1 h at 37°C. Cells were then incubated (30 min, on ice) with anti-mouse monoclonal antibody TLR2 (1 μg/mL; eBioscience, San Diego, CA, USA) or isotype control IgG (eBioscience), washed, and stained (30 min, on ice) with a FITC-conjugated anti-mouse IgG antibody (Jackson Laboratories). After washing and adherence (15 min, room temperature) on cover glass-bottom confocal dishes, the cells were fixed with paraformaldehyde for 15 min at room temperature and mounted (Prolong Gold antifade reagent; Invitrogen). The cell nuclei were stained with DAPI (1:500 dilution in PBS) (Sigma-Aldrich). Cells were imaged with the Leica TCS SP5 confocal system (Leica Microsystems Ltd.). Images were processed and fluorescence was quantified with LAS AF software. In addition, to generate a fluorescence histogram profile, a line was drawn along the cell surface. Fluorescence intensities higher than 40 arbitrary units (isotype control staining) were considered clusters of TLR2 molecules.
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