Goat anti rabbit igg alexa 647

Cells were washed in 0.1% PBS / Tween 20 for 40 min. Samples were blocked with 2% Pig Serum (PS) and 0.5% Triton X-100 in a humidity chamber for 1 h 40 min, then washed twice for 2 min. Samples were incubated with the primary antibody for myosin 1G, previously described [14 (link)] at room temperature overnight at a 1:100 dilution. After five washes with 0.1% PS in PBS/Tween 20, samples were incubated for 1 h at room temperature with goat anti-rabbit IgG Alexa 647 (Jackson ImmunoResearch) at 1:200 dilution; finally, cells were washed twice in PBS/Tween 20. Coverslips were mounted on glass slides using vectashield mounting medium with DAPI (Vector Labs).
To image the stained cells, we used an Olympus FV1000 confocal microscope. We captured no less than 5 micrographs of the median plane of the cells at 60X using identical settings for each capture, making sure that no saturated pixels were detected. We quantified fluorescence intensity from 50 to 100 cells per patient at diagnosis and in the different phases of treatment (remission and consolidation). Images were analyzed using Fiji, Image J software (NIH); specifically, we draw a line in the periphery of each cell to measure the mean fluorescence intensity (MFI) in each cell. Results were expressed as the average of MFI for each patient using arbitrary units for the quantification.