Paraffin-embedded sections from primary tumor of 102 patients with lymph node metastasis were deparaffinized in xylene and hydrated in decreasing concentrations of ethyl alcohol. The sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity, and heated for 10 min at 105 °C by autoclave in Target Retrieval Solution (DAKO, Carpinteria, CA, USA). Nonspecific binding was blocked by incubating with 10% normal rabbit serum for 10 min. The specimens were incubated with anti-GLUT1 antibodies (sc-377228; 1:150; Santa Cruz, Dallas, TX, USA; RRID: AB_2716767) for 30 min at room temperature, anti-PKM2 antibodies (sc-365684; 1:200; Santa Cruz; RRID: AB_10844484) at 4 °C overnight, anti-PDH-E1α antibodies (sc-377092; 1:100; Santa Cruz; RRID: AB_2716767)35 (link) at 4 °C overnight, and with CA9 antibodies (NB100–417; 1:1000; Novus Biologicals, Centennial, CO, USA; RRID: AB_10003398) for 30 min at room temperature. These sections were incubated with a mouse linker for 10 min and peroxidase-labeled polymer (Histofine SAB-PO(M) kit, Nichirei Biosciences Inc., Tokyo, Japan) for 5 min, followed by counterstaining with Mayer’s hematoxylin.
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