Immunofluorescence Staining of Myeloma Cells

Myeloma cells were fixed and permeabilized using the Nuclear Factor Fixation and Permeabilization Buffer Set (Biolegend, San Diego CA), stained with DAPI (20 µg/ml), antibodies against FABP5 (MA5-2402911215, 1.25 µg/mL, ThermoFisher), and Alexa Fluor 647 anti-rabbit secondary antibody (A-21244, 1.25 µg/mL, ThermoFisher). Cells were then rinsed twice with PBS and imaged on a Leica SP5X laser scanning confocal microscope (Leica Microsystems, Buffalo Grove, IL) with Leica LAS acquisition software, using settings as previously described (Fairfield et al., 2021 (link)) using a 20×dry objective on 1.5 mm glass-bottomed dishes (MatTek Corporation, Ashland, MA).

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Farrell M., Fairfield H., Karam M., D'Amico A., Murphy C.S., Falank C., Pistofidi R.S., Cao A., Marinac C.R., Dragon J.A., McGuinness L., Gartner C.G., Iorio R.D., Jachimowicz E., DeMambro V., Vary C, & Reagan M.R. (2023). Targeting the fatty acid binding proteins disrupts multiple myeloma cell cycle progression and MYC signaling. eLife, 12, e81184.

Publication 2023

Nuclear Factor Fixation and Permeabilization Buffer Set Alexa Fluor 647 A-21244 Leica SP5X laser scanning confocal microscope

Alexa fluor 647 Anti antibody Antibodies Buffalo Buffer Cells Confocal laser scanning microscope Dapi Dishes Myeloma Rabbit

Corresponding Organization :

Other organizations : Maine Medical Center, MaineHealth, Tufts University, University of Maine, Dana-Farber Cancer Institute, Harvard University, University of Vermont, University of New England

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Variable analysis

independent variables

Fixation and permeabilization of myeloma cells using the Nuclear Factor Fixation and Permeabilization Buffer Set
Staining with DAPI (20 µg/ml)
Staining with antibodies against FABP5 (MA5-2402911215, 1.25 µg/mL, ThermoFisher)
Staining with Alexa Fluor 647 anti-rabbit secondary antibody (A-21244, 1.25 µg/mL, ThermoFisher)

dependent variables

Cellular localization and expression of FABP5 in myeloma cells

control variables

Use of a 20×dry objective on 1.5 mm glass-bottomed dishes (MatTek Corporation, Ashland, MA)
Imaging on a Leica SP5X laser scanning confocal microscope (Leica Microsystems, Buffalo Grove, IL) with Leica LAS acquisition software, using settings as previously described (Fairfield et al., 2021)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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