Anti human pdcd4 rabbit monoclonal igg

Paraffin sections were stained with anti-human PDCD4 rabbit monoclonal IgG (1 : 100; Cell Signaling Technology) by incubating sections overnight at 4 °C. Secondary staining was performed with biotinylated anti-rabbit IgG at room temperature for 30 min, and tertiary staining was carried out with HRP-conjugated streptavidin according to established protocols. Images were captured using a Nikon microscope imaging system (Tokyo, Japan). Staining intensity was quantified using the Image-pro Plus 6.0 software (Media Cybernetics, Inc., Silver Spring, MD, USA) and expressed as IOD/area.

Anti-human PDCD4 rabbit monoclonal IgG (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), anti-human Akt rabbit monoclonal IgG (1 : 1000; Cell Signaling Technology), and anti-human phospho-Akt (p-Akt) rabbit monoclonal IgG (1 : 1000; Cell Signaling Technology) were used as primary antibodies. Tissues and cellular protein extracts were prepared using RIPA buffer in the presence of a proteinase inhibitor cocktail. We separated 30–40-ng protein extracts by 10% SDS–PAGE and then transferred them onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Protein expression was assessed using Alpha Innotech imaging software (San Leandro, CA, USA).