In Situ Detection of Apoptosis
Nuclei exhibiting DNA fragmentation were detected by in situ hybridization using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Frozen sections were air-dried and fixed in 10% neutral buffered formalin for 10 min. They were then rinsed in two changes of PBS for 5 min each before being postfixed in ethanol:acetic acid (2:1) for 5 min at -20°C. After postfixing, sections were rinsed again in two changes of PBS and then quenched in 3% H2O2 in PBS for 5 min at room temperature. Sections were rinsed in PBS, and equilibration buffer from the kit was applied to cover each section; the buffer was blotted and replaced with a solution of terminal deoxynucleotide transferase (TdT). Cheap Diskus Advair
The sections were incubated with this enzyme for 1 h at 37°C to allow for tagging of 3′ DNA ends with digoxigenin residues. The sections were next placed in stop buffer for 10 min, then rinsed with PBS and incubated with anti-digoxigenin antibody conjugated with peroxidase for 30 min at room temperature. After rinsing in PBS, sections were incubated for 6 min with diamino-benzidine substrate, washed in three changes of distilled water for 1 min each, and then washed in distilled water for 5 min. The sections were counterstained with methyl green for 10 min; this was followed by three washes in distilled water, three washes in 100% butanol, and three washes in xylene before mounting. Negative controls, in which the TdT enzyme solution was replaced with water, were run in every batch of slides stained; this replacement completely eliminated staining. Atretic follicles in whole ovarian sections served as a positive control for apoptotic nuclei. Whole ovarian sections containing these follicles were run in every batch of slides stained.