Alexa 647 labeled goat anti rabbit antibody

Exponentially growing ovarian cancer cells were seeded on sterilized cover glasses (Paul Marienfeld, Lauda-Königshofen, Germany). After exposure to 0.25% stripped FBS-containing medium for 2 days, ovarian cancer cells were treated with 10 μM resveratrol, 10 -7 M T 4 or their combination for 24 h. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min and then permeabilized in 0.06% Triton X-100 for 30 min. Cells were incubated with a polyclonal goatanti-COX-2 antibody or monoclonal rabbit anti-PD-L1 antibody, followed by an Alexa-488-labeled goat antirabbit antibody, Alexa-488-labeled donkey anti-goat antibody or Alexa-647-labeled goat anti-rabbit antibody (Abcam) and mounted in EverBrite Hardset mounting medium with DAPI (Biotium, Fremont, CA). and reverse 5′-GGAGTGGGTGTCGCTGTTG-3′ (accession no.: NM_002046). The real-time PCRs were performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories) using the following conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C. Data calculations of relative gene expressions (normalized to GAPDH reference gene) were performed according to the ΔCT method. Fidelity of the PCR was determined by a melting temperature analysis.