Alexa fluor 555 labeled goat anti rabbit igg

LCC2 and LCC9 cells after transfection were grown on coverslips (174950, Thermanox, Thermo Fisher) and were further treated with 10 μM tamoxifen for 72 hours. Then, the cells were fixed, permeabilized in 0.1% Triton X-100 and blocked in 1% BSA. Then, the cover slips were incubated with antibodies against cleaved caspase-9 at Asp315 (1: 500, PA5-17913, Thermo Fisher) at 4°C overnight. After washing, the coverslips were incubated in a secondary Alexa Fluor-555-labeled goat anti-rabbit IgG (#4413, Cell Signaling) for 30 minutes at room temperature in the dark. Cover slips were mounted with mounting media (ab104139, Abcam), which contained DAPI to stain the nuclei. The number of positive stained cells were scored by counting three sets of at least 100 cells under the microscope.

The carcinoma tissues collected the groups were fixed, embedded into paraffin, and cut into 4-μm-thick sections. The sections were transferred onto cover slips. The sections were dewaxed and rehydrated in Trilogy buffer (Cell Marque, Rocklin, CA) in a pressure cooker for 15 min, blocked in 5% serum for 30 min, and then incubated with antibodies against cleaved caspase-3 at Asp175 (1: 500, #P42574, Cell Signaling, Danvers, MA) at 4°C overnight. After washing, the sections were incubated in a secondary Alexa Fluor-555-labeled goat anti-rabbit IgG (#4413, Cell Signaling) for 30 min at room temperature in the dark. Cover slips were mounted with mounting media containing DAPI to stain the nuclei. The number of cleaved caspase-3-positive cells was scored by counting 3 sets of at least 100 tumor cells each under the microscope.