Is there section deformation resulting in differential change of nuclear numerical densities along the z axis of thick methacrylate or paraffin sections?

The optical disector, a three-dimensional counting frame or probe in stereology, is often positioned in the middle (depth) of a thick section for unbiased nuclear counting. Using 30-40 μm thick methacrylate or paraffin sections for nuclear counting of neurons with the optical disector, however, some studies showed markedly higher nuclear densities at 10% of the section thickness near the top or bottom surface of the section, suggestive of deformation of section along its z axis and thus affecting the number estimation. To verify the findings, this study obtained two sets of 12-14 methacrylate sections (average thicknesses 21.7 and 29.4 μm) and two sets of 12 paraffin sections (average thicknesses 13.8 and 29.2 μm) from mature rat testes. Each section was used to count round spermatid nuclei in the seminiferous epithelium densely packed with the cells, using 3-4 consecutive disectors placed vertically (along the z axis of the section) from the top surface of the section, through the whole section thickness (two sets of methacrylate and paraffin sections) or in 80-83% of the thickness (other sections). The results demonstrated that, overall, there were no considerable nonuniform changes of the nuclear densities along the z axis of the sections.