Figure 2.

Podocyte nuclear mean caliper diameter D direct measurements. In the upper panel, the caliper diameter (cd) for a randomly orientated asymmetric object is the distance between the edges of the object in any single dimension as shown by the calipers (brackets). (A–D) The images show part of a human glomerulus in a 20-μm–thick section of human kidney developed using TLE-4 antibodies to identify podocyte nuclei by red fluorescence. A and B show the top and bottom optical sections respectively of a z-stacked composite image shown in C and D. C shows the z-stack composite in the red channel only. D shows the same z-stack composite using the merged red (TLE-4), green (nonspecific autofluorescence), and blue (DAPI) channels to identify podocyte nuclei (shocking pink) and exclude nonspecific autofluorescence of erythrocytes (orange/green). Podocyte nuclei that are sharply in focus in the top (A) or bottom (B) optical sections are excluded from further analysis, leaving only those podocyte nuclei that cannot have been partially sectioned (shown by yellow stars) to be evaluated. The podocyte nuclear caliper diameter (D) of the starred nuclei can thereby be directly measured by tracing their outer boarders using calibrated imaging software program to estimate the mean caliper diameter of 100 consecutive nuclei. Scale bar, 32 μm.