Imagepro3d software analyses

For immunofluorescent staining, isolated adult cardiomyocytes were fixed for 30 min, room temperature with 2%PFA/PBS/0.1%Triton x-100. Samples were then stained with antibodies for Lamin A/C (A gift from Larry Gerace), Emerin (Santa Cruz), Sun2 (Sigma), α-actinin (Sigma) or Nesprin 2(Abgent). Samples were then stained with secondary antibodies conjugated to CY3 (Jackson ImmunoResearch Inc.). Samples were then incubated with DAPI (Invitrogen) and Phalloidin Alexa-488 (Invitrogen). Samples then imaged using standard confocal microscopy (Olympus FV1000 Confocal). Image analyses were then performed and samples quantified using either Velocity high performance 3D imaging software (PerkinElmer) or ImagePro3D software analyses (MediaCybernetics).

For frozen sections, hearts were fresh frozen using isopentane into frozen blocks (1∶1,OCT∶20%sucrose) and sections cut (7–10 µm). Frozen sections were then post fixed and samples stained first for α-Actinin (Abcam) and then for apoptosis using the In Situ Cell Death Detection kit (Roche) as per manufactures instructions. Images were taken using confocal microscopy (Olympus FV1000 Confocal) and images analyzed using ImagePro3D software analyses (MediaCybernetics).