Alkaline phosphatase labeled antibody

To verify the injection sites, coronal sections (18 μm) were cut on a cryostat (Leica, Germany). Synthesis of riboprobes and in situ hybridization (ISH) was performed as described previously [11] (link). Briefly, sections were fixed in 4% paraformaldehyde (10 m) and acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine (10 m). After prehybridization (2 h) in hybridization solution (containing 50% deionized formamide, 5XSSC, Denhardt's solution, 250 μg/ml tRNA Baker's yeast and 500 μg/ml sonificated salmon sperm DNA), 150 μl hybridization mixture with 400 ng/ml digoxigenin labeled riboprobe against full-length eGFP mRNA (DQ768212) was applied to each slide and slides were incubated overnight at 68°C. Subsequently, the slides were quickly washed in 2XSSC, followed by a 2 h wash in 0.2XSSC, both at 68°C, followed by a 1 h incubation in 10% fetal calf serum in 0.1 M Tris pH 7.5/0.15 M NaCl. Digoxigenin was detected by an alkaline phosphatase labeled antibody (1∶5000; Roche, Germany) using nitroblue tetrazolium and bromochloroindolylphosphate as a substrate. Finally, sections were dehydrated in ethanol, cleared in xylene and mounted with Entallan (Merck, Germany). Pictures of the injection sites were digitalized using a MCID microscope (Zeiss, Germany).

For the in situ hybridization (ISH), cryostat sections of 20 µm thickness from fresh frozen brains were mounted onto slides. Brains were sliced from Interaural: 7.28 mm, Bregma: −1.72 mm to Interaural: 5.64 mm, Bregma: −3.36 mm and the VMH was localized using the atlas ‘The Rat Brain’ (Paxinos and Watson, 6^th edition). Sections were fixed in 4% paraformaldehyde (PFA) for 20 minutes, washed in phosphate buffered saline (PBS), acetylated for 10 minutes and washed again. Sections were pre hybridized in hybridization solution (50% formamide, 5× SSC, 5× Denhardts, 250 µg/ml tRNA Baker's yeast, 500 µg/ml sonicated salmon sperm DNA) for 2 hours at room temperature. The hybridization solution containing 400 ng/ml 720 bp long digoxigenin (DIG)-labeled enhanced green fluorescent protein (eGFP) riboprobe (antisense to NCBI gene DQ768212) was then applied to the slides followed by overnight incubation at 68°C. After a quick wash in 68°C pre warmed 2×SSC, slides were transferred to 68°C pre warmed 0.2×SSC for 2 hours. DIG was detected with an alkaline phosphatase labeled antibody (1∶5000, Roche, Germany) after overnight incubation at room temperature using NBT/BCIP as a substrate. Sections were dehydrated in ethanol, cleared in xylene and embedded in Entellan.
ImageJ Software was used to quantify the spread of GFP mRNA expression in the VMH.