Tissue microarray panel (BCN962, Biomax, Rockville, MD, USA) containing multiple organ carcinoma and adjacent normal tissue was deparaffinized and rehydrated according to standard procedures. Antigen retrieval was performed by heating the slide in 10 mM sodium citrate buffer (pH 6) in a pressure cooker. The highest pressure was maintained for 3 min, and samples were let to cool down for 20 min. Endogenous peroxidase activity was inactivated by incubating samples in 6% H2O2 for 15 min.
Samples were blocked with 20% goat serum in PBS and then incubated ON with primary antibody against PEPCK-M (ab70359, Abcam) and peroxidase-based secondary anti-goat antibody. Antigen-antibody complexes were detected with a DAB peroxidase substrate kit (Dako Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. Samples were counterstained with hematoxylin, dehydrated, and mounted with DPX. Fluorescent preparations were visualized, and images were captured with Nikon Eclipse 800 light microscope (Nikon, Tokyo, Japan).
Samples were blocked with 20% goat serum in PBS and then incubated ON with primary antibody against PEPCK-M (ab70359, Abcam) and peroxidase-based secondary anti-goat antibody. Antigen-antibody complexes were detected with a DAB peroxidase substrate kit (Dako Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. Samples were counterstained with hematoxylin, dehydrated, and mounted with DPX. Fluorescent preparations were visualized, and images were captured with Nikon Eclipse 800 light microscope (Nikon, Tokyo, Japan).