Cleaved caspase-3 staining assay.

Parotid tissue sections were incubated at 37°C for 45 min and rehydrated in histoclear (National Diagnostics, Atlanta, GA) with alcohol gradations [100, 95, 70, and 50% (vol/vol) and water] for 10 min each. Slides were microwaved in citric acid (0.01 M, pH 6.8) twice for 5 min and then cooled for 20 min for antigen retrieval. Slides were washed in phosphate-buffered saline (PBS) for 15 min, blocked with ABC Rabbit Kit (catalog no. PK-6101; Vector Laboratories, Burlingame, CA) for 20 min at room temperature and then incubated overnight at 4°C in a 1:100 dilution of anti-cleaved caspase-3 primary antibody (stock no. 9661; Cell Signaling Technology, Danvers, MA). Then, slides were washed in PBS three times for 10 min, in 1% (vol/vol) H₂O₂ (Fisher Scientific, Fair Lawn, NJ) for 5 min to neutralize endogenous peroxidase activity, in PBS twice for 5 min, and then in biotinylated secondary antibody for 50 min at room temperature per the manufacturer’s instructions (ABC Rabbit Kit; Vector Laboratories). Finally, slides were washed in PBS three times for 5 min, incubated in ABC Reaction Kit (ABC Rabbit Kit; Vector Laboratories) for 30 min, washed in PBS three times for 5 min each, incubated in DAB (Biogenex Laboratories, Fremont, CA) for 6–8 min to develop positive staining, washed in water to stop the reaction, counterstained with hematoxylin for ~2 s (Sigma-Aldrich, St. Louis, MO), rinsed in water for 10 min, dehydrated in alcohol gradations [50, 70, 95, and 100% (vol/vol)] and histoclear for 10 min each, and mounted with Protocol SecureMount (Fisher Scientific, Kalamazoo, MI). Images were taken using a Leica DM5500 and 4-megapixel Pursuit camera. Cleaved caspase-3-positive cells in parotid sections were manually counted from five images per slide at ×400 magnification with four mice per treatment group and quantified by averaging the number of positive cells out of the total number of cells from five fields of view per mouse. Graphs depict the average number of positive cells/total number of cells.