Cells were grown on 2-well glass chamber slides (Ibidi) overnight. Cells were washed with PBS, fixed in 2% paraformaldehyde for 10 min, permeabilized with 1% TritonX-100 for 10 min and blocked with 1% bovine serum albumin (BSA) for 1 h. Cells were incubated in primary antibody or stained with 200 ng/ml FITC phalloidin (Sigma) in blocking buffer overnight on rocker at 4 °C. Cells were subsequently washed 2x in PBS, incubated in conjugated secondary antibody for 2 h on the rocking plate at RT (this step was omitted during phalloidin staining), shaken dry, and counterstained with Fluoroshield with DAPI (Sigma). Cells were imaged on an Olympus IX51 microscope (Olympus) with a 40 x or 60 x oil immersion lens with a UC50 digital camera using cellSense software (Olympus) at identical exposure times. The following antibodies were used: fascin 2 (NOVUS) and α(E)-catenin (GeneTex), Goat-anti-mouse FITC conjugate (Sigma), Goat-anti-rabbit TRITC conjugate (Sigma), Goat-anti-mouse TRITC conjugate (Sigma).
Goat anti mouse fitc conjugate
Manufactured by Merck Group
Sourced in United States
Goat-anti-mouse FITC conjugate is a laboratory reagent used for the detection and identification of mouse-derived proteins or cells in various applications. It consists of goat-derived antibodies that specifically bind to mouse immunoglobulins, conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate). This conjugate allows for the visualization and localization of mouse-derived targets in samples through fluorescence microscopy or flow cytometry.
Automatically generated - may contain errors
2 protocols using goat anti mouse fitc conjugate
1
Immunofluorescence Staining of Cultured Cells
2
Binding Affinity Analysis of BiTE Fragments
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Induced Ramos-Fcε cells were harvested and incubated with BiTE fragments at the concentration corresponding to saturation concentration for staining, in RPMI with 10% FCS and 2 mM L-glutamine, sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 200,000 cells/well, on ice and at 37 °C for 2 h, in triplicates. Antibody fragments were diluted to 300 nM (omalizumab), 35 nM (8D6), 10 nM (ligelizumab and MEDI4212), 200 nM (quilizumab), and 20 nM (blinatumomab). The samples were then placed on ice for 5 min, resuspended in 10 µg/mL anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO, USA) in 2% BSA-PBS, and incubated on ice for 30 min. Binding of anti-FLAG was detected with goat anti-mouse-FITC conjugate (Sigma-Aldrich, St. Louis, MO, USA), diluted 1:200 in 2% BSA-PBS after a 30-min-incubation on ice. The cells were resuspended in 200 µL ice-cold PBS and analyzed with Guava® EasyCyte™ Flow Cytometer (Luminex, Austin, TX, USA).
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