To measure tissue injury (cell death), placental sections were subjected to TUNEL assay using the In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s protocol (Apostolov et al., 2009 (link)). Ki67 antibody (Ab) (Abcam, Cambridge, MA) was used on formalin fixed paraffin embedded placental and embryonic tissues and countered with goat anti-rabbit IgG-Alexa Fluor 594 (Invitrogen) to detect the primary Ab (Apostolov et al., 2009 (link)). Control staining was performed in the absence of the primary Ab. After staining, sections and cells were counterstained with 4′,6-diamidino-2-phenylindol (DAPI) to visualize cell nuclei, mounted under cover slips with Prolong® Antifade kit (Invitrogen, Carlsbad, CA) and acquired using the Olympus IX-81 inverted microscope (Olympus America, Center Valley, PA) equipped with Hamamatsu ORCA-ER monochrome camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan).
The quantification of the TUNEL and Ki67 data iwas performed by using SlideBook 4.2 software on images captured at 200X magnification which counts the nuclei (DAPI staining) and the TUNEL and Ki67 positive cells and calculates the percentage of positive cells to the total number of cells. For Ki67 assessment, quantification by mean intensity was performed to confirm the results.
The quantification of the TUNEL and Ki67 data iwas performed by using SlideBook 4.2 software on images captured at 200X magnification which counts the nuclei (DAPI staining) and the TUNEL and Ki67 positive cells and calculates the percentage of positive cells to the total number of cells. For Ki67 assessment, quantification by mean intensity was performed to confirm the results.
