Monoclonal mouse anti cardiac troponin t

Fixed cells or sections were washed with PBS, permeabilized with 0.1% Triton-X (Sigma-Aldrich, St. Louis, MO), and blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) for 2 hours. Primary and secondary antibodies were applied for 1 hour each, with 3 × 10 minute PBS washes between. Antibody details are as follows: polyclonal rabbit anti-active caspase 3 (1:40, Abcam, Cambridge, MA), monoclonal mouse anti-cardiac troponin T (4 μg/mL, Developmental Studies Hybridoma Bank, Iowa City, IA, Alexa Fluor 488 goat anti-mouse IgG (1:500, ThermoFisher Scientific, Waltham, MA), Alexa Fluor 488 goat anti-rabbit IgG (1:500, ThermoFisher Scientific, Waltham, MA), Alexa Fluor 594 goat anti-mouse IgG (1:500, ThermoFisher Scientific, Waltham, MA). TUNEL staining was conducted with the in situ cell death detection kit (Sigma-Aldrich, St. Louis, MO). Cells and sections were counterstained with DAPI (ThermoFisher Scientific, Waltham, MA) or Alexa Fluor 594 conjugated Wheat Germ Agglutinin (ThermoFisher Scientific, Waltham, MA) per manufacturer protocol. Sections were mounted using ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific, Waltham, MA). Fluorescence images were taken using the Olympus IX81 microscope (Tokyo, Japan) and the Hamamatsu C4742-95 camera (Hamamatsu, Japan). All images were post-processed and quantified using ImageJ.