Hi plan

A Leica DM750 microscope with a manual XY-stage, equipped with a Lumenera Infinity 2-1RC CCD camera (1,392 × 1,040, 4.65 μm square pixels) and a 100W HBO mercury lamp, was used for observation. A Leica Hi Plan 20× (NA 0.40) or 40× (NA 0.65) objective is used to observe and image the cells. Images of at least 100 cells are acquired with DAPI (excitation 350/50 nm, dichroic mirror 400 nm, emission >420 nm) and TRITC (excitation 540/25 nm, dichroic mirror 570 nm, emission >590 nm) filters sets using the Infinity Capture software. The nuclei in both channels are counted by hand, or using an automated method, i.e., by segmentation and “particle analysis” in ImageJ to calculate the percentage of cells that are positive for Cy3 fluorescence, reflecting cells in the S-phase.

For the quantitative assessment of immunostaining, three nonoverlapping images were randomly captured per section using a Leica DML B2/11888111 microscope equipped with a Leica DFC450 camera and Leica C PLAN 10 × 0.22 objective or a Leica DM5500 B/11888817/12 microscope equipped with a Leica DFC450C camera and Leica HI PLAN 10 × 0.25 objective. For each image, the field of view was at 10 × magnification. The number of immunopositive cells in the fields from at least three sections/animal was counted using ImageJ software (US National Institutes of Health, Bethesda, MD, USA) and averaged per field for each animal. This was blindly performed by an independent, experienced researcher. The calculated percentages for at least eight animals per experimental group were considered for comparison and statistical analyses.