Rabbit anti cxcr4 primary antibody

The protein expression and distribution of SDF-1 and CXCR4 in the MI area were assessed by Western blot and IHC. Animals were sacrificed after seven days of treatment and the hearts were harvested quickly.
Subsets of the hearts were used for Western blot (described before), the rest of them were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4-μm thickness for IHC. The antibodies used in IHC were as follows: rabbit anti-SDF-1 primary antibody (1 : 50, Santa Cruz), rabbit anti-CXCR4 primary antibody (1 : 50, Abcam), and the goat anti-rabbit IgG (1 : 100, Beyotime) secondary antibody.

The whole cell proteins were isolated by lysing tissue samples stored at −80°C and CC cell lines in RIPA buffer [100 mM NaCl, 50 mM Tris-Cl (pH 7.4), 2 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1% NP-40, and 0.1% SDS] containing protease inhibitor cocktail (Sigma, USA) on ice. Protein concentration was measured by the Bradford assay. Equal amount of protein (50 μg) was separated using 10% SDS-PAGE and transferred to PVDF membrane (Millipore Corporation, Billerica, MA, USA). Membrane was blocked with 5% nonfat milk in TBST buffer (Tris-Cl, NaCl, Tween-20) for one hour at room temperature and probed with rabbit anti-CXCR4 primary antibody (Abcam, USA) in 5% nonfat milk in TBST overnight at 4°C. After washing with TBST, membrane was incubated with ALP-conjugated goat anti-rabbit IgG (Bangalore Genie, India) in 5% nonfat milk in TBST. After washing twice with TBST, the blot was developed with NBT/BCIP solution (Amresco, USA) and imaged. The membrane was stripped and reprobed with anti-GAPDH antibody (Imgenex, India) as a loading control. All of the immunoblot experiment was repeated twice.

Cell membrane proteins were isolated using a membrane protein extraction kit according to the manufacturer’s instructions (Beyotime, China). Briefly, cells were incubated with lysis buffer at 4 °C for 30 min and then centrifuged for 30 min at 12,000 rpm at 4 °C. Protein concentration was determined by bicinchoninic acid (BCA) protein assay kit. Denatured proteins (20 mg) were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with skim milk dissolved in TBST at room temperature for 2 h and then incubated overnight at room temperature with rabbit anti-CXCR4 primary antibody at a 1:500 dilution (Abcam, UK) and finally incubated with goat anti-rabbit HRP-conjugated secondary antibody at a 1:1000 dilution (Beyotime, China) for 1 h the next day. CXCR4 was normalized to GAPDH (Jian Cheng, China). We quantitatively analyze the signal of protein band by chemiluminescence image processing system (tanon-5200 multi, China). The relative levels of CXCR4 are denoted by the ratio of CXCR4/GAPDH. SDF-1 was normalized to ACTIN (Jian Cheng, China). The relative levels of SDF-1 are denoted by the ratio of CXCR4/ACTIN.