Anti hif 1α

Human biopsy specimens from subjects with several tumor types, including astrocytoma, oligodendroglioma, ependymoma or GBM, were de-paraffinized, rehydrated, and subjected to antigen retrieval in low pH Target Retrieval Solution (Dako) at 95°C for 20 min. Sections were blocked with 5% BSA for 4 h at room temperature, incubated with anti-TFF3 (1:100, Abcam, USA) and anti-HIF-1α (1:100, Abcam, USA) antibodies overnight at 4°C. Cultured cells were fixed with 4% paraformaldehyde for 20 min and incubated with anti-TFF3 (1:100, Abcam, USA) and anti-HIF-1α (1:100, Abcam, USA) antibodies overnight at 4°C. Sections were stained with Alexa Fluor 488- and 647-conjugated secondary IgG (1:500, Abcam) for 1 h at room temperature. Images were acquired with an FV1000 Laser Scanning Confocal Microscope (Olympus, Japan).

To identify HIF-1α- and HIF-2α-expressing cells by cytometry, cells were detached with a scraper and collected with cold PBS. Cells were then fixed with 4% paraformaldehyde (PFA) at pH 7.4 for 15 min at 4 °C and 5 min at room temperature (RT). Cells were permeabilized with 0.3% Triton X-100 for 15 min on ice and blocked with 5% FBS at 4 °C for 30 min. Cells were incubated at 4 °C for 1h with anti-HIF-1α 1:100 (Abcam), anti-HIF-2α 1:100 (Abcam), anti-HIF-1α (OH P402) 1:100 (Abcam), and anti-NANOG 1:500 (Cell Signaling). Cells were then washed 3 times in PBS and incubated with anti-mouse Alexa fluor 488 1:500 (Invitrogen,), anti-rabbit Alexa fluor 633 1:500 (Invitrogen,), and anti-rabbit IgG-PE 1:500 (Abcam) for 30 min at RT. Cells were then washed 3 times at 4 °C with PBS and resuspended in 500 mL of PBS. Fluorescence analysis was carried out by flow cytometry (FACSCalibur; BD Biosciences). In this assay, we use as an isotype control the following antibodies: anti-rabbit IgG phycoerythrin (PE) goat isotype (Abcam); rabbit control IgG (Abcam), and mouse IgG1 isotype control mAb (Cell Signaling). The percentage of positive HIF-1α and Hif-2α cells was calculated by FACSCalibur software after we selected a correct cell gate. The program analyzes the percentage of positive HIF-1α and Hif-2α cells.

The relative protein levels of renalase and HIF-1α were analysed using Western blot analysis similar to what was described previously 17 (link),18 (link). The primary antibodies, anti-renalase, anti-HIF-1α, anti-β actin and anti-GAPDH were from Abcam (goat anti-renalase polyclonal antibody, ab31291, 1:500 dilution, for HK2 Western), (rabbit anti-renalase monoclonal antibody, 1:500 dilution, for rat tissues Western), Novus Biologicals (Littleton, CO, USA) (NB100-105, mouse anti-HIF-1α monoclonal antibody, 1:500 dilution), Sigma-Aldrich (A5441, mouse anti-β-actin monoclonal antibody, 1:10,000 dilution) and Santa Cruz (sc-48166, goat anti-GAPDH polyclonal antibody, 1:5000 dilution) respectively. The secondary antibodies were from Santa Cruz (horseradish peroxidase-conjugated anti-rabbit and anti-goat IgG) or Sigma-Aldrich (horseradish peroxidase-conjugated antimouse IgG). GAPDH and β-actin were used as internal control for renalase and HIF-1α respectively. All the data were obtained from ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA) and band intensity was analysed using Image Lab 4.0.1 software.