Mouse anti hif 1α monoclonal antibody

Protein extracts (20 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 4-12 % gel, transferred to nitrocellulose membranes, and blocked for 1 hour at room temperature in Blocking One (Nacalai Tesque, Tokyo, Japan). The membranes were incubated with rabbit anti-ADORA2B polyclonal antibody (Chemicon International, Temecula, CA, USA), mouse anti-α tubulin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti- HIF-1α monoclonal antibody (Santa Cruz Biotechnology), rabbit anti-Akt monoclonal antibody (Santa Cruz Biotechnology), rabbit anti-pAkt monoclonal antibody (Santa Cruz Biotechnology) , rabbit anti-ERK1/2 monoclonal antibody (Santa Cruz Biotechnology), and rabbit anti-pERK1/2 monoclonal antibody (Santa Cruz Biotechnology) overnight at 4 °C. The membranes were washed with 0.1 % Tween-20 in Tris-buffered saline, incubated with secondary antibody and coupled to horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega, Madison, WI, USA) for 1 hour at room temperature. The membranes were detected using SuperSignal West Pico Chemiluminescent substrate (Thermo), and immunoblotting was visualized by exposing the membranes to ATTO Light-Capture II (ATTO, Tokyo, Japan). Signal intensities were quantitated using the CS Analyzer version 3.0 software (ATTO).

All paraffin-embedded tissues were dewaxed, treated with 10 mM sodium citrate for heat-induced antigen recovery, incubated with 0.3% H₂O₂ for 20 minutes at room temperature, and then co-cultured with 20% normal goat serum for 30 minutes. Immunohistochemistry was performed using mouse anti-HIF-1α monoclonal antibody (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted at 1:100 in blocking reagent at 4℃ overnight. The slides were continuously cultivated with 1:200 horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) (Chemicon International, Inc., Temecula, California, USA) for two hours at room temperature. They were then developed with diaminobenzidine for 8-12 minutes, counterstained with hematoxylin, dried, and then fixed with resin. The images were obtained using a cell image analysis system and medical image analysis software, and the data were quantitatively analyzed. Three separate visual fields were randomly selected from each slice to obtain the percentage of positive area in each sample (positive cell area/total area) and to be used for analysis. The results were assessed by two experienced pathologists who were blinded to the samples.