The signal obtained after such a background correction was considered to be an antigen-specific signal. For each image, specific antibody staining was merged with nuclear staining (blue) using Soft Imaging System software (Soft Imaging System Corp., Lakewood, CO) and MetaMorph 4.6 software (Universal Imaging Corp., Downington, PA) that caused virtually no pixel shifting during image merger and resulted in shades of red, green, and blue.
To verify the reproducibility of the data, immunofluorescence localization studies were repeated at least three times per serially sectioned ovary using rat ovarian tissues from different animals (n = 15), porcine oocytes (n = 30), zygotes (n = 40), and blastocysts (n = 20 of different quality). Representative photomicrographs were arranged using Adobe PhotoShop (Adobe, San Jose, CA) without any further adjustment to maintain the true nature of the findings.
In Situ Localization of Apoptotic Cells: TUNEL
Ovaries fixed in 10% neutral buffered formalin were dehydrated through a graded series of ethanol, cleared in xylene, embedded in paraffin, and sectioned (section thickness, 4 |xm). Sections mounted on positively charged slides (ProbeOn Plus; Fisher Scientific, Pittsburgh, PA) were deparaffinized, hydrated, and treated with 20 |xg/ml of proteinase K (37°C, 30 min), and endogenous peroxidase activity was removed by treatment with 3.0% hydrogen peroxide (room temperature [RT], 10 min).