4.4. Immunofluorescence and co-localization analysis

Cells were seeded at a density of 1 × 10⁵ glass⁻¹ cover slip, 24 h before treatment. Following treatment (hypoxia/DMOG), cells were rinsed three times with phosphate-buffered saline (PBS; Sigma Aldrich, MO, USA) and subsequently fixed for 15 min with 4% paraformaldehyde (Sigma Aldrich, MO, USA) at room temperature, followed by three 10 min PBS washes. In total, 50 mM NH₄CL was added for 20 min, removed and the cells were blocked for 20 mins (blocking buffer: 1% BSA (Sigma Aldrich, MO, USA), 0.1% Triton X-100 (Sigma Aldrich, MO, USA) and 0.4% Tween 20 (Sigma Aldrich, MO, USA) in PBS). Cells were then incubated for 1 h with primary antibody diluted in blocking buffer at room temperature. Cells were washed three times in blocking buffer and incubated for 30 min at room temperature with the secondary antibody (diluted in blocking buffer). The following antibodies were used: rabbit anti-HIF-2α (1 : 500; #ab20654/1 : 100; #ab179825, Abcam, Cambridge, UK), mouse anti-HIF-1α (1 : 1000; #610959, BD Biosciences, CA, USA), mouse anti-HIF-1β (1 : 100; #NB100-124, Novus Biological, CO, USA), mouse anti-RNAPII phospho ser5 (1 : 50; #ab24759, Abcam, Cambridge, UK), mouse anti-SC35 (1 : 1000; #ab11826, Abcam, Cambridge, UK), mouse anti-Sart1 (10 µg ml⁻¹; #ab88583, Abcam, Cambridge, UK), anti-rabbit Alexa Fluor 555 (1 : 1000; #A-21428, Invitrogen, CA, USA) and anti-mouse Alexa Fluor 488 (1 : 500, #A-11008, Invitrogen, CA, USA). Topro-3 Iodide (diluted 1 : 1000 in PBS; Invitrogen, CA, USA) was used to stain the nuclei. Samples were imaged with a Plan-Fluar 40×/NA 1.30 oil immersion objective on a LSM 710 confocal microscope (Zeiss, Oberkochen, Germany). Alexa Fluor 488 was excited with 488 nm Argon laser, Alexa Fluor 555 was excited with 561 nm HeNe1 laser and Topro-3 Iodide was excited with 633 nm laser. Images were captured using Zen 2010 software (Zeiss, Oberkochen, Germany).

Post-acquisition processing was carried out using ImageJ [47]. The middle slice from each z-stack was analysed. The background was subtracted for both the red and green channel (pixel size of 5 for HIF-2α, 5 for RNAPII, 10 for HDAC5-YFP). Analysis was performed with an ImageJ plugin for co-localization analysis (http://fiji.sc/Colocalization_Threshold).