Immunofluorescence of 15-LOX and 5-LOX in Macrophages

Immunofluorescence microscopy was carried out with slight modifications of procedures reported in (12 (link)). M2 macrophages (1 × 10⁴ cells) were seeded on gelatin-coated glass coverslips in a 12-well plate and incubated in normoxia or hypoxia for 24 hours before fixation with 4% paraformaldehyde (15 min) and staining. Cells were then fixed with 4% paraformaldehyde (15 min) followed by incubation in 0.25% Triton X-100 (Sigma-Aldrich) containing Background Sniper (10 min, room temperature) to reduce background staining. Cells were then incubated with rhodamine phalloidin (Life Technologies) (30 min) for actin visualization, followed by incubation with mouse monoclonal anti-15-LOX (1:100) and rabbit monoclonal anti–5-LOX (1:100) (Abcam) overnight at 4°C. Coverslips were washed and labeled with the following secondary antibodies: Alexa Fluor 488 donkey anti-mouse (1:1000) and Alexa Fluor 647 donkey anti-rabbit (1:1000) (Life Technologies). To visualize the nuclei, coverslips were mounted with VECTASHIELD (Vector Laboratories) and examined by Zeiss LSM 800 with an Airyscan confocal system on a Zeiss Axio Observer Z1 inverted microscope equipped with an Axiocam 503 Monochrome Camera.