Zoe fluorescent cell imager microscope

To assess the effects on cell death, we performed a TUNEL assay (terminal deoxynucleotidyl transferase [TDT]-mediated dUTP Nick end labeling) on cells or on fixed paraffin-embedded left ventricular (LV) sections, using a commercial kit (Roche). The assay was performed according to the manufacturer's instructions. After staining, the sections were incubated with the DAPI fluorescent dye (Sigma-Aldrich). Next, 5-6 images were acquired for each sample using ZOE Fluorescent Cell Imager Microscope (Bio-Rad Laboratories). Then, the percentage of apoptotic cells was calculated using the ImageJ software, by evaluating the percentage of cells colored with DAPI and positive to TUNEL.

Forty-eight hours after transfection, cells were washed twice with cold PBS, fixed for 30 min with 4% paraformaldehyde, washed thrice with PBS, and permeabilized for 10 min with 0.2% Triton X-100. They were then washed thrice with PBS, blocked in 3% BSA for 30 min at 37°C, and washed 3 more times with PBS. Mouse anti-HA tag (1:500) and mouse anti-human GBP1 (1:1,000) monoclonal antibodies were used as primary antibodies. Both primary antibodies were co-incubated for 1 h at room temperature. After 3 washes with PBS, fluorescein-isothiocyanate-labeled goat anti-mouse IgG (1:500) was added and cells were incubated for 50 min (protected from light). All antibodies were diluted in PBS containing 3% BSA. The cells were washed 6 times with PBS, and an anti-fluorescence-quencher was added to each well. Transfected cells were examined under a ZOE Fluorescent Cell Imager microscope (Bio-Rad, Hercules, CA).