4 6 diamidino 2 phenyindole dilactate dapi

CTRL and Pt primary fibroblasts were cultured onto 24 × 24 mm glass slides in six-well plates and incubated for 30 min at 37 °C, with the following probes: 2 µM Fluo-4 AM (Life Technologies, Carlsbad, CA, USA) for cytosolic Ca²⁺ and 4 µM X-Rhod-1 AM (Invitrogen, Molecular probes^TM, Carlsbad, CA, USA) for mitochondrial Ca²⁺ level measurements. Stained cells were washed with Phosphate Buffered Saline (PBS), fixed in PBS containing 4% paraformaldehyde for 10 min at room temperature, and cell nuclei were stained with 1 μg/mL 4′,6-Diamidino-2-phenyindole, dilactate (DAPI) Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Stained cells were examined with a Leica TCS SP8 microscope (images collected using a 40× and 60× in oil immersion objective) coupled to the Laser Scanning Confocal Microscopy (LSCM) system. Acquisition, storage, and data analysis were performed using Leica software LAS AF 3 (https://www.leica-microsystems.com/products/microscope-software/).

HUVECs were grown on 14 mm glass coverslips (4 × 10⁴ cells/well) placed in 24 well plates. Cells were treated with 100 ng/mL recombinant TNF-α or 20% sera from preeclamptic women in the presence or absence of 1 μg/mL hydroxychloroquine for 16–22 h prior to fixing with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature. The treatment groups were compared with untreated HUVECs or cells treated with 20% normal pregnancy sera. Cells were blocked with 0.5% bovine serum albumin (BSA, Sigma-Aldrich) for 30 min, incubated first with rabbit anti-ZO-1 (1:50, Zymed) overnight at 4°C, then with donkey anti-rabbit Alexa Fluor 568 (1:100, Invitrogen) for 1 h in the dark. Cell nuclei were stained with 2 μm 4′,6-diamidino-2-phenyindole dilactate (DAPI, Sigma Aldrich) for 10 min and mounted with fluorescent mounting media (DakoCytomation). Staining was examined with an Olympus BX60 fluorescent microscope and images were taken using an Olympus DP70 camera and Olympus CellSens software (Olympus). The primary antibody was replaced with an isotype-matched control antibody in the negative controls. The mean intensity of the staining was assessed using ImageJ software (version 2.0.0-rc-43/1.50i, http://imagej.net/Fiji/Downloads, Bethesda, MD).