Kinetic of annexin fusion-tagged protein translocation to plasmalemma was monitor in live cells or fixed trophoblasts using a laser scanning confocal microscope (SP5; Leica) equipped with a 63x N.A. 1.2 water-immersion objective in the line-scan mode. Trophoblasts were incubated with ionomycin (10 μM). For live cells, images were acquired before incubation and automatically within 20 s intervals for 10 min after incubation.
Optical recording of intracellular Ca2+ transient was performed by loaded trophoblasts with the membrane-permeant Fluo-4 AM as recommended by the manufacturer protocol. Wide-field images were obtained with Olympus BX50WI upright microscope with a 40 × 0.8 NA water-immersion objective and an ORCA-AG camera (Hamamatsu)50 (link). Images were acquired automatically within 60 s intervals. Pseudo-color images display the intracellular Ca2+ value coded in hue and the fluorescence intensity coded in intensity.
Optical recording of intracellular Ca2+ transient was performed by loaded trophoblasts with the membrane-permeant Fluo-4 AM as recommended by the manufacturer protocol. Wide-field images were obtained with Olympus BX50WI upright microscope with a 40 × 0.8 NA water-immersion objective and an ORCA-AG camera (Hamamatsu)50 (link). Images were acquired automatically within 60 s intervals. Pseudo-color images display the intracellular Ca2+ value coded in hue and the fluorescence intensity coded in intensity.
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