Secondary goat anti rabbit antibody

The paraffin-embedded slides were routinely deparaffinized and hydrated. The slides were then retrieved in citric acid buffer (pH 6.0), heated in a microwave oven for 10 min, and then treated with 3% H₂O₂ for 15 min and washed with Tris-buffered saline containing 1% Tween 20 (TBST) three times each for 5 min. After a 60 min incubation in normal goat serum (Boster, Wuhan, China), the slides were incubated with primary antibodies overnight at 4°C. The antibodies included polyclonal rabbit anti-BRD7 (1:500 dilution), polyclonal rabbit anti-LDHA (1:2000 dilution) from Proteintech (Wuhan, China), polyclonal rabbit polyclonal rabbit anti-Cyclin D1 (1:200 dilution) from Santa Cruz (Dallas, TX, USA), polyclonal rabbit anti-c-PARP (1:200 dilution) and polyclonal rabbit anti-p21 (1:500 dilution) from Cell Signaling Technology (Danvers, MA), and polyclonal rabbit anti-Ki67 (1:200 dilution) from Bioworld Technology, Inc. (St. Louis, Minnesota, USA). The slides were washed three times with TBST (each for 15 min) and then incubated with a secondary goat anti-rabbit antibody (Maixin, Fujian, China) for 60 min at 37 °C. Then, the slides were visualized with 3,3′-diaminobenzidine (DAB) (Zhongshan Gold Bridge, Beijing, China) for 5 min and counterstained with hematoxylin for 45 s. The slides were mounted and photographed with an Olympus BX51 microscope (Olympus, Japan).

The paraffin-embedded slides were routinely deparaffinized and hydrated. The slides then were retrieved in citric acid buffer (pH6.0) heated by microwave for 20 min. The slides then were treated with 3% H₂O₂ for 15 min and washed by tris-buffered saline containing 0.1% Tween 20 (TBST) three times each for 5 min. After 60 min incubation in normal goat serum (Boster, Wuhan, China), slides were incubated with primary antibody (polyclone rabbit anti-BRD7 (1:500 dilution) from Proteintech, Wuhan, China; polyclonal rabbit anti-p53 (1:500 dilution), anti-phosphorylated MDM2 at ser166 (pMDM2, 1:1000 dilution), anti-c-PARP (1:200 dilution) and anti-p21 (1:200 dilution) from Cell Signaling Technology, Danvers, MA; polyclonal rabbit anti-Ki67 (1:200 dilution) from Bioworld Technology, Inc. St. Louis, Minnesota, USA) overnight at 4 °C. The slides were washed three times with TBST (each for 15 min) and then incubated with a secondary goat anti-rabbit antibody (Maixin, Fujian, China) for 60 min at 37 °C. Then the slides were visualized with 3,3'-diaminobenzidine (DAB) (Zhongshan Gold bridge, Beijing, China) for 5 min and counterstained with haematoxylin for 45 seconds. The slides were mounted and photographed with Olympus BX51 microscope (Olympus, Japan).