Blocking agent

Tumor tissues were fixed with 10% neutral-buffered formalin for 24 h and embedded in paraffin. Paraffin-embedded 5-μm sections were deparaffinized and rehydrated, washed in distilled water, and subjected to heat-mediated antigen retrieval. Endogenous peroxidase activity was quenched by treatment with 3% H₂O₂ for 30 min, and sections were washed with phosphate-buffered saline for 5 min. The sections were blocked for 1 h with a blocking agent (Invitrogen) and incubated overnight with antibodies against LDHB or PDH (1:1000, Cell Signaling) at 4 °C. The sections were then incubated at room temperature with a biotinylated secondary antibody (1:500, Vector Laboratories, Burlingame, CA, USA) for 1 h. The slides were washed and treated with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories). Sections were then counterstained with hematoxylin and mounted in aqua mount (Vector Laboratories).

The tumor tissues were fixed with 10% neutral buffered formalin and embedded in paraffin for sectioning. Tissue sections (5 μm thick) were cut from the paraffin-embedded blocks using a microtome. The sectioned tissues were incubated at 60 °C for 2 h and subsequently deparaffinized in xylene and dehydrated in a graded ethanol series. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in distilled water for 30 min. Heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) in a microwave oven, followed by washing thrice with PBS. The sections were blocked using a blocking agent (Invitrogen, Frederick, MD, USA) and incubated overnight with the primary antibodies at 4 °C. The primary antibodies used were as follows: EGFR (1:100, Santa Cruz Biotechnology) and Src (1:100, Santa Cruz Biotechnology). The sections were washed with PBST and incubated with biotinylated anti-mouse antibody (1:500, Vector Laboratories, Burlingame, CA, USA) for 1 h. After a PBST wash, the sections were treated with 3,3′-diaminobenzidine substrate (Dako, Carpentaria, CA, USA) and counterstained with hematoxylin (Thermo Fisher Scientific, Waltham, MA, USA). A Leica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany) was used for image analysis.