Sp5 x mp inverted laser scanning confocal microscope

Cardiac tissues were incubated with 10 μM di-8-ANEPPS (Invitrogen, Carlsbad, CA) for 15 min at 37°C to stain the cell membrane. Tissues were then rinsed with normal Tyrode’s solution (1.8 mM CaCl₂, 5 mM glucose, 5 mM HEPES, 1 mM MgCl₂, 5.4 mM KCl, 135 mM NaCl, 0.33 mM NaH₂PO₄, pH 7.4) (Sigma Aldrich, St. Louis, MO) and imaged at 37°C using a Leica SP5 X MP Inverted Laser Scanning Confocal Microscope (Leica, Wetzlar, Germany) with a 63x glycerin objective. The cell borders were traced manually in ImageJ (NIH, Bethesda, MD), the cell area was then measured by fitting the traced area to an ellipse, and the aspect ratio was determined using the ratio of the major axis (length of cell) to the minor axis (width of cell).

Fixed cardiac tissues were incubated with Cell Mask® (Invitrogen, Carlsbad, CA) for 10 min. The tissues were rinsed three times with PBS (Invitrogen, Carlsbad, CA), mounted and sealed as described above, and imaged on a Leica SP5 X MP Inverted Laser Scanning Confocal Microscope (Leica, Wetzlar, Germany) with a 63x glycerin objective. A total of 9 fields of view were imaged per sample. Images were rotated 90° in the x direction and confocal stacks were converted into 2D images using a sum z-projection. The resulting image was then subjected to a threshold, converted to a binary image, and tissue thickness was measured by outlining the tissue layer to obtain width measurements using ImageJ (NIH, Bethesda, MD).