Rabbit anti cxcr4 antibody

Brain sections (30 μm in thickness) were rinsed in phosphate-buffered saline (PBS). Endogenous peroxidase activity was quenched by washing the sections in a solution of PBS with 3% H₂O₂ and 10% methanol for 15 minutes. After subsequent rinsing in PBS, blocking was achieved by incubation in a solution containing 5% normal donkey serum (NDS) in 0.25% Triton X-100 in PBS (Tx/PBS) for 1 hour. Thereafter, sections were incubated with a mouse anti-neuronal Nuclei (NeuN) antibody (diluted at 1:1000, Merck Millipore, Billerica, MA, USA) or a rabbit anti-CXCR4 antibody (diluted at 1:200; Abcam, Cambridge, UK) at 4°C in 5% NDS in Tx/PBS overnight. The next day, sections were rinsed in 1% NDS in Tx/PBS followed by incubation with respective biotinylated secondary antibodies in 2% NDS in Tx/PBS at room temperature for 90 minutes. Sections were washed with Tx/PBS and incubated with avidin-biotin-complex (ABC; Vector Laboratories, Burlingame, CA, USA) for 1 hour. Following this incubation, the sections were rinsed in PBS. The ABC reaction was visualized using NiDAB (Dabsafe, Saveen Werner AB, Limhamn, Sweden) with the addition of NiCl₂ and a mixture of 3% H₂O₂ in H₂O. Thereafter, sections were rinsed in PBS, mounted on glass slides, dried overnight, dehydrated, treated with xylene and then cover-slipped with Pertex (Histolab AB, Västra Frölunda, Sweden).