Color camera digital sight ds u1

Tissues were surgically removed, fixed with 4% (wt/vol) paraformaldehyde, paraffin‐embedded, and sectioned. Sections were deparaffinized by xylene and re‐hydrated in graded alcohol. Slides were preincubated with blocking solution (Kretz et al, 2013), 2% (wt/vol) BSA, 2% (vol/vol) normal goat serum, and 0.02% Tween 20 in Tris‐buffered saline (pH 7.4) and then stained with primary antibodies overnight at 4°C. Slides were incubated with secondary antibody (HRP rabbit or mouse antibody; DAKO EnVision System, Agilent, Cat # K4065) for 30 min at room temperature. After washing, sections were incubated in peroxidase substrate solution (DAB; DAKO, Agilent, Cat # GV825), rinsed in water, and counterstained with hematoxylin. Immunohistochemistry was performed with anti‐Ki‐67 (Cat #14‐5698‐82), anti‐cleaved Caspase 3 (Abcam, Cat # ab2302) antibodies. Images were acquired with OLYMPUS BX51 up‐right microscope (UPIanAPO 40×/0.85 objective lenses) connected to Nikon color Camera Digital Sight DS‐U1 and analyzed with the NIS‐elements software. Randomly taken images (at least five fields per animal) were captured from tissue sections and processed with ImageJ software (National Institutes of Health).