Dm4 up light microscopy

Cells undergoing apoptosis were quantitatively assessed using a TUNEL assay kit (DeadEnd^TM, Promega, Madison, WI, USA). Deparaffinized tissue sections prepared as described above were sequentially treated with the reagents contained in the kit according to the protocols provided by the manufacturer. At the end of this procedure, coverslips were mounted on a sliding glass and visualized by DM4 up-light microscopy (Leica, Wetzlar, Germany). Under a fluorescence illumination setting (excitation/emission wavelength: 395/509 nm), three HFPs (400×) per rat were randomly chosen and the TUNEL-positive cells were averaged across groups.

Deparaffinized tissue sections prepared as described above were reacted with a tissue retrieval solution for 5 min at 120 °C, after which their endogenous peroxidase activity was blocked using 1% hydrogen peroxide for 1 h at 22 °C. Following three PBS washes, slides were incubated with rabbit Cleaved caspase-3 antibody (Cell signaling, Denver, MA, USA) diluted in PBS (1:200) in a humid chamber for 24 h at 4 °C. After PBS washes, the slides were incubated with biotinylated anti-rabbit IgG (Vector, Burlingame, CA, USA) diluted in PBS (1:250) for 2 h at 22 °C. Information regarding product number of the antibodies used in this study are presented in supplementary material (Table S1). After washes in PBS, the slides were reacted with a VECTASTAIN-Elite avidin-biotin complex Kit (Vector, Burlingame, CA, USA) for 30 min at 22 °C. In order to visualize the immunoreactivities, the slides were reacted with the chromogen 3,3’-Diaminobenzidine (DAB) (Vector, Burlingame, CA, USA) for 30 min at 22 °C. The three HFPs (400×) per rat were randomly chosen and visualized by DM4 up-light microscopy (Leica, Wetzlar, Germany). The number of the cleaved caspase-3-immunoreactive cells (dark-brown colored) was averaged across groups.