Dako aqueous mount

Paraffin-embedded sections were cleared using boric acid solution by microwave boiling for 15 min to retrieve antigens. Sections were blocked with 3% H₂O₂ in methanol for 10 min, washed, and then blocked with endogenous peroxidase. After washing, sections were blocked with 3% bovine serum albumin for 1 h at RT. Staining was performed at 4°C overnight with the indicated antibodies along with anti-EHMT2 (R&D Systems, 1:100), followed by incubation with biotinylated secondary antibodies (BioGenex) for 40 min at RT. Finally, sections were incubated with Strep-ABC complex (Dako, K-0377) for 30 min at RT. Sections were developed using a AEC substrate kit (Vector lab, SK-4200) for 20 min at RT, counterstained with hematoxylin, dried, and mounted using a DAKO aqueous mount (Dako, 003181).

Staining of human tissues was performed as described (31). The paraffin embedded sections were cleared and the sections were incubated with 0.1% Pronase (Roche #165 921) in 0.1% CaCl2 pH 7.8. at 37C for 10 minutes. They were blocked with 3% H2O2 in TBS for 10 mins., washed then blocked with Dako Biotin Blocking System (Dako X0590). After washing, they were further blocked with 10% Normal Rabbit Serum for 10 mins at room temperature (RT) and incubated firstly with p-ERK (Abcam, ab65142) dilution 1:50, PKM2 (Abcam, ab38237) dilution 1:100 and GLUT-1 (Abcam, ab652) dilution 1: 100for 1hr at RT, then with biotinylated Rabbit Anti-Mouse (Dako, E-0354) at 1/100 for 30 mins. at RT, and finally with Strep-ABC complex (Dako, K-0377) at 1/100 for 30 mins. at RT. The sections were developed with AEC substrate kit (vector lab, SK-4200) at RT for 20 mins., counterstained with haematoxylin and mounted with DAKO aqueous mount (Dako, 003181).

The paraffin embedded sections were cleared and the sections were incubated with 0.1% Pronase (Roche #165 921) in 0.1% CaCl2 pH 7.8. at 37C for 10 minutes. They were blocked with 3% H2O2 in TBS for 10 mins., washed then blocked with Dako Biotin Blocking System (Dako X0590). After washing, they were further blocked with 10% Normal Rabbit Serum for 10 mins at room temperature (RT) and incubated firstly with PDK1 (Abcam, ab110025) dilution 1:50, and GLUT-1 (Abcam, ab652) dilution 1: 1000 for 1h at RT, then with biotinylated Rabbit Anti-Mouse (Dako, E-0354) at 1/100 for 30 mins. at RT, and finally with Strep-ABC complex (Dako, K-0377) at 1/100 for 30 mins. at RT. The sections were developed with AEC substrate kit (vector lab, SK-4200) at RT for 20 mins., counterstained with haematoxylin and mounted with DAKO aqueous mount (Dako, 003181).