Effects of different fixatives on Delta, FWHM, and the apparent mechanical properties of kinetochores. (A) Control and taxol-treated metaphase RPE1 cells antibody immunostained for CenpT (red) and Hec1 (green) after fixation with glutaraldehyde (left) or formaldehyde (right). Location of the centrioles (Centrin-GFP) is marked by blue arrows. Notice that the chromosomes (visualized with Hoechst 33342) appear slightly swollen after formaldehyde fixation. (B) Delta values calculated by both methods I and II and FWHM values for CenpT and Hec1 are shown for each fixation condition under the corresponding image in A. Note that Delta is consistently lower in formaldehyde-fixed samples. Also notice a highly statistically significant change in FWHM of Hec1 in glutaraldehyde-fixed samples. FWHM of both CenpT and Hec1 are larger after formaldehyde fixation, and the values are not affected by taxol. Asterisks indicate statistical significance of mean value difference between control and taxol-treated cells as assessed by two-tailed t test (*, P < 0.05; *** P, < 0.001; ****, P < 0.0001). (C) Scatterplots of Delta versus interkinetochore distance and the values of kinetochore compliance for each of the four conditions. Meth., method.