Maternal Smoking Highly Affects the Function, Membrane Integrity, and Rheological Properties in Fetal Red Blood Cells

Figure 3

Visualization of the confocal images with varying morphology in relation to the phosphorylation of NOS3 in RBC-NS and RBC-S populations. The representative confocal -stack images show RBC-NS (a–c) and RBC-S (d–f) with regular biconcave and RBC-S echinocyte (g–i) with the echinocyte phenotypes. The panels (a, d, and g) show the RBCs immunolabelled with a mouse primary anti-NOS3 antibody followed by an Alexa Fluor® 647 secondary antibody. The panels (b, e, and h) represent the RBCs immunolabelled with a rabbit anti-p-NOS3 (p-NOS3) primary antibody, followed by an Alexa Fluor® 488 secondary antibody. The microscopic parameter, i.e., the thickness of -axis, was 3.845 μm/slice, and the scale bar of 5 μm was kept constant at all instances. The RBCs were randomly selected from both RBC-NS (-45 regular biconcave-shaped cells from each of 3 independent samples) and RBC-S (-35 regular biconcave cells from each of 5 independent samples and echinocytes from each of 4 independent samples) groups. In the highlighted zone of interest, zooming ratios were 4.486, 6.865, and 10.019 in (a)–(c), (d)–(f), and (g)–(i), respectively. The ImageJ 1.51n software was used to quantify the mean intensity levels from three middle slices in the RBC-NS, RBC-S, and RBC-S echinocyte for NOS3 (j) and p-NOS3 (k). (l) The ratio between NOS3 and p-NOS3 intensity levels. All statistical analyses were accepted by one-way ANOVA using the Newman-Keuls multiple comparison test at , , and . MFI: mean fluorescence intensity.