*2.6. Immunohistochemistry*

LV tissues were first embedded in paraffin; these paraffin blocks were cut into 5-µm-thick sections, which were placed onto slides and washed thrice for 3 min with phosphate buffered saline (PBS) solution and incubated in 3% hydrogen peroxide for 30 min. Afterwards, the slides were washed thrice with PBS and incubated in PBS with 10% goat serum and 1% bovine serum albumin (BSA) at room temperature for 2 h. The sections were incubated overnight at 4 ◦C with anti-cleaved caspase-3 antibodies (1:600; Cell Signaling Technology) diluted in PBS with 1% BSA. The slides were gently washed thrice with PBS and incubated in anti-rabbit secondary antibodies (1:1000; Santa Cruz Biotechnology) at room temperature for 1 h, and washed thrice with PBS. The ABC reagent kit (1:100; Vector Laboratories, Burlingame, CA, USA) was used to amplify the protein signals, and the sections were visualized by staining with 0.03% diaminobenzidine (a chromogenic substrate) at room temperature. The slides were stained and dehydrated via a hematoxylin control method, and then cover-slipped. The cleaved caspase-3–positive cells were quantified in the LV tissue sections using the ImageJ software (version 1.52a; NIH, Bethesda, MD, USA).
