Luteal Regression in the Normally Cycling Rat: MATERIALS AND METHODS(5)

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.


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