*4.9. TUNEL Apoptosis Assay*

The embedded wax block sections were dewaxed in water and treated with proteinase K and rupturing cell membrane working solution at 37 ◦C for 22 min, then washed with PBS at pH 7.4 three times. The mixture of TDT and DUTP (1:5) was incubated at 37 ◦C for 2 h, followed by 4',6-diamidino-2-phenylindole (DAPI) staining for 10 min after washing. The results were observed under a Nikon Eclipse C1 fluorescence microscope with Nikon DS-U3 imaging system (Nikon Corporation, Japan) and the images were collected. At a magnification of 200×, the positive cells of TUNEL staining were counted in 4 random fields on each section.
