2.7. Stereology

Step serial sections of complete explants that were immunohistochemically stained with an antibody to PCNA were quantified by estimating the volume density of PCNA‐positive cells in the sections of control and experimental explants. The analysis was performed under the Nikon Alphaphot binocular light microscope (Nikon, Vienna, Austria) using Weibel's M42 test system, made of 42 short test lines each with two ends as test points.33 Volume density of PCNA‐positive cells was estimated by counting points of the test system, which hit PCNA‐stained nuclei, and points which hit the reference space at the magnification of 400 × (reference space being defined as hits on any part of an explant). Volume density (V _v) of PCNA is the ratio between the hits falling in all PCNA‐stained nuclei (P _i) and hits falling in the reference space (P _t; V _v = P _i/P _t), and is expressed in mm⁰ (mm³/mm³).33, 34 Before analysis, for each experimental group, the stereological orientation measurement was carried out to define the number of fields to be tested.34 At least 90 fields were stereologically assessed.