Hc was incubated at 1X108 yeast/mL with 10% serum and the indicated chelators (10 mM EGTA or EDTA) in PBS for 30 min at 37°C. The yeast/particles were washed 2X with PBS, and co-cultured with BMDMs, or stained with a FITC-conjugated anti-mouse C3 antibody (MP Biomedicals, 1:200) for 1h at RT. Following staining, yeast/zymosan were washed 2X with PBS, and fixed with 4% PFA after spinning onto poly-L-Lysine-coated coverslips. Coverslips were washed 2X and imaged at 60X magnification to visualize mouse C3 deposition on the cell-wall.
Fitc conjugated anti mouse c3 antibody
Manufactured by MP Biomedicals
The FITC-conjugated anti-mouse C3 antibody is a laboratory reagent used for the detection and analysis of the complement component 3 (C3) protein in mouse samples. The antibody is conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate) to enable visualization and quantification of the target protein using techniques such as flow cytometry or immunofluorescence microscopy.
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4 protocols using fitc conjugated anti mouse c3 antibody
1
Yeast-Macrophage C3 Deposition Assay
2
Complement Deposition on Streptococcus pneumoniae
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Bacterial strains grown in TS broth at 37°C to an OD of 1.0 at 620nm were washed and resuspended in 1X PBS. 2.5x106 CFU of Spn in 25 μL were incubated with an equal volume of fresh normal mouse serum at 37 °C for 30 min. For negative controls, serum was pre-treated at 57°C for 30 min to heat-inactivate complement. The incubation was ended by adding 50 μL of 20 mM EDTA in 1X PBS. Cells were pelleted, resuspended, and then stained with FITC-conjugated anti-mouse C3 antibody (MP Biomedicals) at 4°C for 30 min. Cells were then fixed in 4% PFA and resuspended in 20 mM EDTA in 1x PBS. Samples were analyzed by flow cytometry using the LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
3
Immunohistochemical Analysis of Kidney C3 and Annexin A2
4
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.
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