Immunofluorescence staining

Myocardial samples prepared in optimal cutting temperature (OCT) medium were processed for immunofluorescence (IF). From each heart, 10 μm cryostat sections were placed on slides and incubated with 4% paraformaldehyde for 15 min and washed thrice with PBS. Sections were then permeabilized using 0.25% Triton X-100 followed by three washes with PBS. For GSH staining, after permeabilization, the sections were incubated in ethanol containing 10 mM N-Ethylmaleimide for 30 min and washed three times with PBS. This was followed by blocking in 5% normal goat serum in PBS for 1 h and appropriate primary antibodies incubations overnight at 4°C at the following concentrations: mouse anti GSH-NEM (1:500) and rabbit anti-Ubiquitin (1:500). The sections were then incubated with secondary antibody Alexa fluor 488 goat anti-rabbit/ mouse IgG (H+L) (1:1,000) followed by mounting with fluoroshield/DAPI (ab104139, Abcam, Cambridge, MA, USA). Images were captured using Nikon A1Confocal microscope (Nikon Instruments Inc.) at 60X magnification. Fluorescence signals from three different random areas per section were captured, and the intensity was quantified using Image J software (NIH) and expressed as relative fold change with respect to the control group.