Sant sc 1226

Sections containing ovarian stroma were immunostained to measure apoptosis via cleaved-caspase-3 expression (SANT-SC-1226, 1:100, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, EUA) and the terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nickend labeling (TUNEL) assay using a commercially available kit (In Situ Cell Death Detection Kit, Fluorescein, Roche, Berlin, Germany, 11684795910) following the manufacturer’s instructions. Red-brown coloring of the cytoplasm/nucleus of the granulosa cells was considered positive staining (any other coloring was considered negative staining). For the negative controls, the primary antibody was omitted to avoid bias.
Images of the sections were obtained using an image acquisition software system (Leica DM2500); measurements were made using the Leica QWin V3 software. Red-brown coloring of the cytoplasm/nucleus of the stroma cells was specified as positive staining (anything else as negative staining). A positive cell staining assessment was performed in four different fields per animal at 200x magnification and the results are expressed as a percentage of the positive area (arbitrary unity/mm²). Two independent investigators blinded to the experimental treatments performed all measurements.

Sections containing ovarian stroma were immunostained to measure apoptosis via cleaved-caspase-3 expression (SANT-SC-1226, 1:100, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, EUA) and terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nickend labeling (TUNEL) assay using a commercially available kit (In Situ Cell Death Detection Kit, Fluorescein, Roche, Berlin, Germany, 11684795910) following the manufacturer’s instructions. For the negative controls, the primary antibody was omitted to avoid bias.
Images of the sections were obtained using an image acquisition software system (Leica DM2500); measurements were made using the Leica QWin V3 software. Red-brown coloring of the cell cytoplasm/nucleus of the cells was specified as positive staining (any other coloring was considered negative staining). A positive cell staining assessment was performed in four different fields per animal at × 200 magnification, and the results are expressed as a percentage of the positive area (arbitrary unity/mm²). Two investigators blinded to the experimental treatments performed all measurements (LLD and MES). In case of doubt or discordant analysis, a third investigator (JMS) was requested.