After rats were euthanized, TGs were quickly dissected, immersed immediately in liquid nitrogen, and stored at −80°C to determine the abundance of PARP and its products, poly(ADP-ribosyl)ated polymers (pADPr), by Western blot analysis. TGs were homogenized in radioimmunoprecipitation assay buffer (20 mM Tris-HCl, pH 7.5, 0.1% [w/v] sodium lauryl sulfate, 0.5% [w/v] sodium deoxycholate, 135 mM NaCl, 1% [v/v] Triton X-100, 10% [v/v] glycerol, and 2 mM EDTA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Tissue homogenates were centrifuged at 14,000g for 15 minutes, and the supernatant was collected. The total protein concentration was determined by bicinchoninic acid assay protein assay. Lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was placed in blocking buffer (5% nonfat milk in 1× Tris-buffered saline with 0.1% Tween-20 [TBST]) for 1 hour at room temperature (RT) with shaking and then incubated with primary antibodies overnight at 4°C with shaking. Following incubation, the membrane was washed 3 times with TBST and incubated with horseradish-peroxidase-conjugated secondary antibodies in TBST for 1 hour at RT with shaking. After three washes with TBST and soaking with the chemiluminescent reagent, protein bands were detected using a ChemiDoc MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA). ImageJ software (version 1.47; US National Institutes of Health, Bethesda, MD, USA) was used to calculate the signal intensity of the bands. The values were normalized relative to β-actin. The primary antibodies used were rabbit anti-PARP1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-pADPr (Santa Cruz Biotechnology), and rabbit anti-β-actin (Cell Signaling Technology, Danvers, MA, USA).
Some of the collected TGs were used for immunohistochemistry. After overnight fixation in 4% paraformaldehyde (PFA) at 4°C, the tissues were soaked in 30% sucrose for 24 hours and then frozen in optimal cutting temperature compound (Tissue-Tek, Sakura Fine Technical Co., Ltd., Tokyo, Japan) at −80°C. Cryostat sections (10 μm) were mounted on silane-coated slides (Matsunami, Osaka, Japan), which were permeabilized in 0.1 M phosphate-buffered saline with 0.1% Triton-X (PBST) and blocked in 0.1% PBST containing 1% bovine serum albumin and 5% donkey serum for 1 hour at RT. The slides were incubated with anti-PARP and anti-pADPr primary antibodies in 0.1% PBST overnight at 4°C and incubated with secondary antibodies in 0.1% PBST for 2 hours at RT. After being washed three times, slides were mounted with a 4′,6-diamidino-2-phenylindole (DAPI)-supplemented mounting medium and examined using fluorescence microscopy (Axio Observer; Carl Zeiss Meditec, Hamburg, Germany).