Anti rfp antibody

ARE-luc2 mice were sacrificed and perfused transcardially with cold saline solution followed by 4% paraformaldehyde in PBS. Brains were immediately removed, post-fixed in the 4% paraformaldehyde fixative for 24 h and then transferred in solutions of sucrose at increasing concentrations (up to 30%) during the following 72 h. Samples were then frozen and stored at −80°C for successive analyses. Serial coronal sections of 40 μm were cut throughout the brain using a freezing sliding microtome (Leika SM 2000R) and stored at −20°C in a solution containing 30% ethylene glycol, 20% glycerol and 0.05 M sodium phosphate buffer until use. The slide-mounted sections were rinsed in PBS and incubated in PBS containing 10% NGS and 0.3% TX-100 at room temperature. Sections were then incubated at 4°C in PBS/1% NGS/0.3% TX-100 containing a rabbit polyclonal anti-RFP antibody (diluted 1:400; Abcam, Cambridge, UK). After a 24 h incubation sections were rinsed in PBS and incubated 1 h at RT in PBS/1% NGS containing the Alexa Fluor 594 conjugated goat anti-rabbit IgG antibody (1:300; Molecular Probes, Carlsbad, CA, USA). Finally, sections were rinsed in PBS and covered with Prolong with DAPI (Molecular Probes, Carlsbad, CA, USA).