Isolated hAMCs were seeded onto 24-well plates. After adherence, cells were washed three times with PBS and fixed by 4 % paraformaldehyde at room temperature for 30 min. Then, they were blocked with 1 % BSA for another 30 min. Subsequently, cells were incubated overnight at 4 °C with a mouse anti-human vimentin antibody (1:500 dilution, Chemicon, USA) or a mouse anti-human STRO-1 antibody (1:500 dilution, Chemicon, USA) respectively. After three washes, cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, Life Technologies, Grand Island, NY, USA) or FITC-conjugated goat anti-mouse IgM (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Finally, cells were observed under a fluorescent microscope.
Fitc conjugated goat anti mouse igm
Manufactured by Merck Group
Sourced in United States
FITC-conjugated goat anti-mouse IgM is a laboratory reagent used to detect and quantify mouse IgM antibodies in various immunoassays. The reagent consists of goat-derived antibodies specific to mouse IgM that are conjugated to the fluorescent dye fluorescein isothiocyanate (FITC).
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7 protocols using fitc conjugated goat anti mouse igm
1
Immunocytochemical Characterization of hAMCs
2
Immunofluorescence Analysis of Breast Cancer Cells
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Human breast cancer cells MCF-7 and MDA-MB-231 (2.5 × 105) were cultured on glass coverslips, and after 48 h of rBmK AGAP treatment, cells were fixed with 4% paraformaldehyde for 30 min after washing with PBS. Cells were then permeabilized with 0.1% Triton X-100 for 5 min and blocked with complete serum for 30 min at 37°C. The MCF-7 and MDA-MB-231 cells were incubated with primary antibodies of interest at 4°C overnight followed by a FITC-conjugated goat anti-mouse IgM or TRITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich, China) for 1 h at room temperature. The cells on the coverslips were then incubated with DAPI for 10 min at room temperature. Images were obtained under a fluorescence microscope (Olympus BX83, Japan).
3
Flow Cytometric Analysis of IgM Expression
4
Quantification of Oxidized Phospholipids in Choroidal Neovascularization
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Eyes were first fixed in 4% paraformaldehyde for 1 h, and then the corneas and lenses were removed. Eyecups were incubated in 30% sucrose overnight and frozen at −80 °C after immersion in optimal cutting temperature compound (OCT: Tissue Tek; EMS). The eyecups were cut into 12 µm cryosections. Sections were incubated with E06 monoclonal antibody (1:100, Avanti POLAR LIPIDS, Inc., Alabaster, Alabama, Cat # 330001) overnight at 4 °C. After rinsing, the sections were incubated for 1 h with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (1:100, Sigma-Aldrich, St. Louis, MO, Cat # F9259), Alexa Fluor 568-conjugated antibody for isolectin B4 (lectin, 1:200, Invitrogen) to stain the vessels, and Topro 3 (1:1,000, Life Technologies, Grand Island, NY) to stain the nuclei. The sections stained with no primary antibody were used as controls. Labeling for all sections was performed during the same experimental session. Images were captured using confocal microscopy (Olympus IX81) at 20X magnification. Within the sections of lectin-stained CNV, E06 labeling to measure oxidized phospholipids (OxPLs) was determined with fluorescent density using Image J . At least ten CNV lesions per condition were analyzed (n=6 per condition).
5
Serum Antibody Binding Assay
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Sera from recipient mice were decomplemented by heating to 56 °C for 30 min as described previously (3 (link)). Equal amounts of sera and cell suspensions (5 × 106 /mL) were incubated for 45 min at 4 °C. Cells were labeled with FITC-conjugated goat anti-mouse IgM (Sigma-Aldrich) and analyzed by flow cytometry (FACS Calibur, BD Bioscience).
6
Immunofluorescence Staining of Cells
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After washing with PBS, cells plated on cover slips were fixed with 4% paraformaldehyde for 30 min. Cells were then permeabilized with 0.1% Triton X-100 for 5 min. After being blocked with complete serum for 30 min at 37°C, the cells were incubated with primary antibody at 4°C overnight. The cells were then incubated with FITC-conjugated goat anti-mouse IgM or TRITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Images were captured with an Olympus BX83 fluorescence microscope (Japan).
7
Ganglioside GQ1b Surface Levels
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Ganglioside GQ1b levels on the cell membrane was analyzed as described previously [39, 40, 46] . Briefly, A549, MCF7, HCT116 and U87MG cells were transferred to round cover slips (pretreated with 2% 3-aminopropyltriethoxysilan) in an 8-well multi-plate. After incubation with a GQ1b monoclonal antibody (Mouse IgM, Kappa-chain, clone: GMR 13; Seigakagu; Japan) diluted 1:500 with 5% BSA in PBS, cells were labeled with 1:500 FITC-conjugated goat anti-mouse IgM (Sigma; St. Louis, MO, USA) as a secondary antibody. The samples were mounted on slides and viewed using confocal laser-scanning microscope (LSM 510; Zeiss; Jena, Germany).
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