Alexa fluor 568 conjugated anti rabbit antibody

20 μm slices were cut by a cryotome (Thermo, USA). Slices were dried in ventilation for 30 minutes and then permeabilized by 0.25% triton-X 100 for 15 minutes. The sections were blocked with 10% bovine serum albumin (Sigma, USA) in phosphate buffer saline (PBS) diluent for 1 hour at room temperature. Then sections were incubated with rabbit monoclonal anti-CXCR4 (1 : 200, Abcam, USA) or rabbit polyclonal anti-TNF-α (1 : 200, Abcam) overnight at 4°C. After slices were washed with PBS for 10 minutes 3 times, added Alexa Fluor 568-conjugated anti-rabbit antibody (1 : 500, Abcam, USA) diluted in antibody dilution buffer (Dako, Denmark) for 1 h at room temperature and followed by nuclear staining using DAPI (Cell Signaling Technology, USA). Images were detected by a confocal microscope LSM 710 (Zeiss) and measured using ImageJ Software (National Institutes of Health, USA).