Anti pkm1

Consecutive 3-μm sections were cut from each block and subjected to immunohistochemical staining with the EnVision+DualLink system (Dako, Carpinteria, CA, USA). An immunoperoxidase technique was applied following antigen retrieval with microwave treatment (95 °C) in citrate buffer (pH 6.0) for 45 min. Anti-PKM1 (Abgent, San Diego, CA, USA), anti-PKM2 (Cell Signaling Technology, Danvers, MA, USA), HIF1α (Thermo Fischer Scientific, Waltham, MA, USA), and anti-Ki-67 antibody (DAKO) diluted at 0.5 μg/mL were used as primary antibodies. After 2 h of incubation at room temperature, the sections were incubated with a secondary antibody for 30 min. The specimens were color-developed with diaminobenzidine (DAB) solution (Dako) and counterstained with Meyer’s hematoxylin (Sakura Finetek, Tokyo, Japan). Immunostaining of all samples was performed using the same conditions of the antibody reaction and DAB exposure. Immunoreactivities of PKM1 and PKM2 were classified according to the Allred’s score (AS) [50 (link)]: grade 1 for AS = 0, 2 for AS = 2–4, and 3 for AS = 5–8. Grade 2 and 3 cases were regarded as immunopositive [5 (link)]. We also observed 20 microscopic fields per case at 200× magnification and counted the tumor cells per case; the results were expressed as the percentage of tumor cells, with positive nuclei defined by Ki-67 and HIF1α LI [10 (link)].

Whole-cell lysates were obtained using the M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific Inc., Rockford, IL, USA). Each lysate (50 μg) was subjected to 12.5% SDS-PAGE and immunoblotting by electrotransfer to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). The filters were incubated with anti-PKM1 (Abgent) and anti-PKM2 antibodies (Cell Signaling Technology) further amplified with peroxidase-conjugated IgG (MBL, Nagoya, Japan). The immune complex was visualized by the ECL Western blotting detection system (GE Healthcare, Amersham place, UK). An anti-GAPDH antibody (Santa Cruz Biotechnology Santa Cruz, USA) was used as an internal control. The specific bands on the immunoblotted membrane detected by each antibody were quantified with NIH image computer software (National Institutes of Health, Bethesda, MD). The signal intensities were compensated for by GAPDH intensities as internal controls.