Antibody for cleaved caspase 3

Cultures for imaging were fixed at day 10 with 4% paraformaldehyde in PBS for 10 minutes at room temperature before permeation with 0.1% Triton-X for 10 minutes. For myosin heavy chain staining, myotubes were incubated overnight at 4°C with purified mouse IgG against myosin heavy chain type I (1:100, BA-D5, Developmental Studies Hybridoma Bank, deposited by Schiaffino, S.), then with rabbit anti-mouse IgG conjugated with Alexa Fluor 647 (1:500 for 1 hour at room temperature). For endothelial cell identification, FITC-conjugated BS1 (1:500 for 1 hour at room temperature, Sigma-Aldrich) was utilized to stain cells for imaging. Staining for apoptosis was achieved using an antibody for cleaved caspase-3 (Cell Signaling Technology, MA, USA) diluted at 1:200. Antibodies for ceramide (Sigma Aldrich) and CerS2 (Boster Biological Technology, CA, USA) were also used at a 1:200 dilution for staining and imaging. F-actin was stained using Alexa Fluor 488-conjugated phalloidin (Invitrogen, CA, USA). All secondary antibodies were utilized at a 1:500 dilution. Nuclei were stained with DAPI (Invitrogen). Stained cells were then imaged using a Zeiss Axiovert 200 widefield fluorescence microscope. Quantitative analysis of images was accomplished using ImageJ software (National Institutes of Health, MD, USA), and were conducted by two blinded researchers independently.