TUNEL assay. TUNEL was performed as described previously. Briefly, paraffin-embedded, whole-ovarian follicle sections (thickness, 45 |xm) were mounted on positively charged slides, deparaffinized, hydrated, washed thoroughly three times for 5 min each in 1X PBS, and immersed in PBS with 0.3% (v/v) H2O2 (10 min, RT; to inhibit endogenous peroxidase activity). Following three additional 5-min washes in PBS, the sections were incubated (30 min, RT) in a blocking reagent (Large Volume DAKO LSAB Kit; DaKo Diagnostics Canada, Inc., Mississauga, ON, Canada) and immersed in 50 |xl of the TUNEL mixture (47.5 |xl of TUNEL label containing fluorescein isothiocyanate [FITC]-dUTP and 2.5 |xl of TUNEL enzyme) in a humidified chamber (37°C, 60 min). They were mounted for fluorescence microscopy with a confocal laser-scanning system (Bio-Rad 1024). The FITC signal in TUNEL-positive cells was excited at 488 nm, with images collected within the wavelength range of 506-538 nm. allergy medications
DNA fragmentation analysis. Apoptotic cell death was also assessed on the basis of DNA fragmentation and confirmed through visualization of discrete DNA fragments of 185-bp multiples on agarose gel electrophoresis. Follicular DNA was extracted using Qiagen Tissue Amp Kit according to the manufacturer’s instructions. The DNA was quantified spectrophotometrically by the absorbance at 260 nm. The DNA was end-labeled by incubating with TdT and [a-32P]ddATP as previously described. Briefly, 500 ng of DNA sample were added to a mixture (5 |xl of 5X TdT buffer, 2.5 |xl of 10X CoCl2, 0.5 |xl of TdT enzyme, and 0.5 |xl of a 10 mCi/ml concentration of [32P]ddATP and Tris EDTA buffer) to a total volume of 25 |xl and then incubated at 37°C for 60 min. Unincorporated nucleotides were removed with the Qiagen nucleotide removal kit, and the labeled samples were subsequently resolved by 1.8% (w/v) agarose. The gel was dried (3 h) and then exposed to a Bio-Rad Phosphor-Imager, and low-molecular-weight DNA (<4 kilobase pairs) and genomic DNA were densitometrically quantified. The gel was then exposed to x-ray film at — 80°C. To correct for possible uneven gel loading, the ratio of low-molecular-weight DNA (representing apoptosis) to genomic DNA was calculated for each sample, and means of the ratios were compared. The intraobserver variability, determined by performing two separate DNA ladder analyses on the same sample, was approximately 5%.