Anti hif 2α

Immunohistochemistry with DAB detection was performed according to a protocol described previously (Tjoa et al., 2006 (link)). Anti-HIF-1α was from Novus Biologicals (used at 1:250; Cambridge, UK) and anti-HIF-2α was from Abcam (used at 1:250; Cambridge, UK). Both antibodies required heat-induced antigen retrieval (Tris-EDTA buffer, pH 9).

Cells were collected and lysed in lysis buffer on ice. Total proteins were separated by 10 % SDS-PAGE and blotted on PVDF membrane. Membranes were blocked with 10 % non-fat milk powder at room temperature for 2 h and incubated with primary antibodies: anti-HIF2α (1:200), anti-VE-cadherin (1:1000), anti-MMP2 (1:1000), anti-MMP9 (1:5000), anti-Twist1 (1:200), anti-Twist2 (1:50) (all from Abcam, Cambridge, UK) and anti-GAPDH (1:1000, Santa Cruz Biotechnology, CA, USA), at 4̊C overnight. After three washes, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000; Santa Cruz Biotechnology). Reactive bands were detected using ECL western blotting detection reagent (GE Healthcare, USA).

All chemicals were purchased from Sigma Aldrich unless otherwise specified. The following antibodies were used for immunoblotting: anti-pVHL [BD Pharmagen (Ig32; 556347; 1:1,000)], anti-α-tubulin (Sigma Aldrich; T6199; 1:5,000), anti-CHCHD4 (Sigma Aldrich; HPA034688; 1:1,000), anti-HIF-2α (Abcam; Ab199; 1:500), anti-ATP5B (Abcam; ab14730; 1:1,000), anti-mtCO-2 (Abcam; ab110258; 1:1,000), anti-β-actin (Abcam; 6276; 1:5,000), anti-HSP60 [Cell Signaling Technology (CST); #4870; 1:1,000], anti-VDAC1 (Abcam; ab15895;:1,000), anti-COX IV (Abcam; ab14744; 1:1,000), anti-PHB1 (CST; #2426; 1:1,000), anti-anti-GLUT1 (Alpha Diagnostics; GT12-A; 1:1,000), anti-DRP-1 (CST; #5391S; 1:1,000), anti-pDRP-1 (Ser⁶³⁷) (CST; #4867S; 1:1,000), anti-HIF-1α (BD Biosciences; 610959; 1:1,000). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Amersham and used at 1:5,000 dilution. For immunocytochemistry anti-ATP5B (Abcam; ab14730) was used at 1:500 dilution and Alexa Fluor 568 goat anti-mouse IgG (Invitrogen; A-11031) was used at 1:5,000 dilution.

Paraffin sections were stained with HE and then observed by two independent physicians. The number of eosinophils was counted at high power (HP) magnification (400×), and 10 HP fields were randomly selected and analyzed. Referring to the method of Liu et al. [36 (link)], CRSwNP samples were classified as eosinophilic when the percentage of eosinophils exceeded twice the SD of the mean of controls (4.8% + 2×2.12%= 9.04%; therefore, 9% was chosen as the cut-off value).
All monoclonal antibodies (anti-IL-17A, anti-HIF1α, anti-HIF2α, anti-IL-17A receptor, anti-IFN-γ, and anti-TSLP) were purchased from Abcam (Cambridge, MA, USA). An anti-IL-5 polyclonal antibody was purchased from Novus Biologicals. Details of the experimental methods are included in the Supplemental material. Image Pro-Plus 6.0 software was used to analyze images. All images are taken at exactly the same conditions using the same microscope and camera ;image brightness was normalized to background levels. We selected epithelial layer as area of interest (AOI) for measurements. Using Image Pro Plus, the images were changed to grayscale digital images to test total optical density (OD) and calculate the area of AOI; then calculated the mean OD using total OD/area of AOI. The mean OD represents the density of dye staining and reflects the content of the target protein.

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.