Immunofluorescence Analysis of CXCR4, RAC1, and RHOA

The dewaxed sections were incubated in citrate antigen retrieval solution (Biosharp, Hefei, China) at 95–100 °C for about 20 min. After cooling to room temperature, the sections were washed in PBS and blocked with 5% goat serum for 1 h, then incubated for 12 h at 4 °C with primary antibodies: anti-CXCR4 antibody (1:100), anti-RAC1 antibody (1:100), and anti-RHOA antibody (1:100). Next, sections were incubated with FITC-conjugated secondary antibodies (ZF-0311, Zsbio, Beijing, China) for 1 h at room temperature in the dark. Sections were washed three times in the dark with PBS for 3 min each time. Sections were sealed under coverslips using FluoroshieldTM with DAPI (F6057, Sigma-Aldrich, Zwijndrecht, The Netherlands, USA) seals and analyzed using fluorescence microscopy [57 (link)]. Fluorescence intensity was analyzed and quantified by Image J.