Te2000 s inverted fluorescence microscope

Pru-FYVE-mCherry (Pru-FYmC), P∆aat1-FYVE-mCherry (P∆aat1-FYmC), and P∆aat1:AAT1-FYVE-mCherry (P∆aat1:AAT1-FYmC) parasites were collected using the same protocol for esterified amino acid viability assay. After incubation, extracellular parasites were settled on Cell-Tak (Fisher Scientific)-coated slides for 30 min at 4°C, fixed in 4% formaldehyde, and stained with a rabbit anti-mCherry antibody (1:500, Merck) followed by an Alexa 594-conjugated anti-rabbit IgG antibody (1:1,000). Three hundred images for each sample were acquired by focusing on the PLVAC signal on a Nikon TE2000-S Inverted Fluorescence Microscope equipped with a ×100 oil objective and processed using ImageJ software. PLVAC size was measured by defining the area of anti-mCherry immunofluorescence.

Twenty four hours after OGD, cardiomyocyte monolayer was fixed and stained with Hoechst 33342 (Sigma) to quantify apoptotic cells. The morphological features of apoptosis (cell shrinkage, chromatin condensation, and fragmentation) were monitored by Nikon Optical TE2000-S inverted fluorescence microscope. At least 400 cells from 12 randomly selected fields per well were evaluated to determine the number and percentage of cells exhibiting apoptosis, and each treatment was performed in duplicate.