Alexa fluor 488 594 labeled second antibody

The bladders and kidneys were aseptically harvested, and embedded in OCT compound with liquid nitrogen. Frozen sections (5 μm) were cut and air-dried at room temperature for 20 min and fixed with cold acetone for 10 min. Then the tissues were immediately submerged into methanol for 20 min and 3% hydrogen peroxide in methanol for 10 min. After rehydration in PBS, sections were blocked with 5% BSA for 1 h, incubated with F4/80 antibody (Abcam, ab6640, 1:100), CD36 antibody (Proteintech, 18836-1-AP, 1:100) in blocking buffer overnight at 4°C. After that, coverslips were washed five times with PBS, and incubated with FITC-labeled secondary antibody (Proteintech) or Alexa Fluor 488/594-labeled second antibody (Proteintech) for 1 h. Finally tissue sections were counterstained with DAPI for nuclei visualization. Images were acquired using the OLYMPUS IX73 fluorescent microscope (Shinjuku, Tokyo, Japan).

Cells were grown on a Lab-Tek chambered coverglass, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 10 min and blocked with PBS containing 10% goat serum for 1 h at room temperature. Samples were incubated with primary antibody (E. coli LPS antibody, Abcam, ab35654, 1:200; C/EBPα antibody, Cell Signaling Technology, 8178, 1:200; LXRβ antibody, Abcam, ab28479, 1:200; CD36 antibody; Proteintech, 18836-1-AP, 1:200) in PBS containing 10% goat serum at 4°C overnight, washed five times with PBS, incubated with Alexa Fluor 488/594-labeled second antibody (Proteintech) at room temperature for 1 h. Then 100 nM rhodamine phalloidin was added and incubated at room temperature for 30 min, and DAPI was added for 10 min. Cells were imaged using a confocal fluorescence microscope (FV1000-D, Olympus, Tokyo, Japan).