4.6. Immunofluorescence Staining and the Quantification of the Cardiomyocyte Size

iPSC-derived cardiomyocytes were initially fixed in the 4% paraformaldehyde solution and permeabilized in 0.1% Triton X-100 solution, followed by the blocking of non-specific binding with the PBS buffer containing 5% normal goat serum. Then, these cells were incubated with primary antibodies using the indicated antibodies and conditions described in Supplemental Table S2. After washing of the cells with PBS for three times, these cells were then incubated with the goat anti-mouse secondary antibodies conjugated with either FITC (green) or PE (red). DAPI (blue) was used for the staining of nucleus. A laser-scanning confocal microscope (Olympus) was used for the imaging of labeled cells and the amount of retained autofluorescent materials were measured in the red (546) and green (488) channels by the quantification of the pixel areas (Adobe Photoshop/Image J software (version 1.50d, National Institutes of Health, Bethesda, MD, USA). The antibodies used for the immunofluorescence staining are listed in the Supplemental Table S2.

For the measurement of cardiomyocyte size, the size was evaluated by measuring the cellular area contents of iPSC-derived cardiomyocytes generated from either normal subjects or patients with Fabry cardiomyopathy. Twenty days after cardiac induction, the spontaneously beating embryoid bodies were dissociated into single cells using Accutase solution (Stem Cell Technology). These cells were then plated onto gelatin-coated dishes for further experiments and analysis. Subsequently, the cellular images of cTnT-positive cells were recorded at 30, 40 and 60 days post-induction using the confocal microscope (FV10i, Olympus). The area of cTnT-positive cells was measured and the cellular area pixels were analyzed using the ImageJ software package (NIH). About fifty cells were analyzed in three independent experiments.