Histochem Cell Biol (2012) 137:11–24 diethyl pyrocarbonate (DEPC)-treated water. In vitro transcription was performed according to the manufac- turer’s instructions for 2–3 h at 37 C in a water bath using Biotin-, Fluorescein- or Digoxigenin (DIG)-RNA labeling mix (all Roche, Mannheim, Germany) and the appropriate RNA polymerases (Fermentas, Leon-Rot, Germany). For single labelings, cRNAs were labeled using Biotin-, Fluo- rescein- or DIG-RNA labeling mixes. For double labelings, both Fluorescein- and DIG-labeled cRNA probes were used for NPY and 5-HT2C mRNA detection as required, for 5-HT1A mRNA detection only DIG-labeled cRNA was used. For triple labelings, NPY cRNA was labeled using Biotin-RNA labeling mix, 5-HT2C cRNA with Fluores- cein-RNA labeling mix and 5-HT1A cRNA with DIG- RNA labeling mix. After in vitro transcription, template DNA was removed by adding 20 U DNase I (Roche), incubation was carried out for 15 min at 37 C in a water bath and aliquots were run on an agarose gel (see above) to control success of in vitro transcription. Then, samples were puriﬁed with phenol/chloroform extraction (using water-saturated, stabilized phenol, AppliChem, Darmstadt, Germany) followed by precipitation as mentioned above. Air-dried pellets were dissolved in DEPC-treated water and concentration and riboprobe quality was assessed spectro- photometrically. Probes can be stored at -20 C for several months. To make sure that the different cRNA probes used in multiple ISH do not cross-react, we aligned the cRNA sequences of interest before conducting ISH experiments using ‘‘MultAlin’’, a program for multiple sequence alignment (Corpet 1988). Animals and tissue preparation For the subsequent experiments, brains of 3-month-old Wistar rats purchased from Charles River (Sulzfeld, Germany) were used. Animal experiments were carried out according to the German Law for the Protection of Animals, and were designed to minimize number and suffering of animals used. Anaesthetized animals were decapitated, the brains were dissected and cut into coronal slices of approximately 5-mm thickness. The slices were immediately placed in Tissue Freezing Medium (Leica Microsystems, Wetzlar, Germany) on cork supports, snap frozen in liquid nitrogen-cooled 2-methylbutane and stored at -80 C until use. 10-lm-thick serial coronal sections containing hippocampus and amygdala (Bregma -2.12 to -3.3 mm; Paxinos and Watson 2007) were cut on a cryostat set at -25 C and thaw-mounted on Superfrost Plus microscopic slides (Menzel, Braunschweig, Germany). Afterwards, sections were ﬁxed for 5 min in freshly pre- pared 4% paraformaldehyde (PFA) in 0.01 M phosphate- buffered saline (PBS) at 4 C, transferred to chilled ethanol and stored at 4 C until usage. 13 In situ hybridization If not mentioned otherwise, all working steps were per- formed at room temperature. Rehydration and washing were carried out by placing the microscopic slides in glass jars containing approximately 60 ml of the appropriate solutions. If heating of solutions was required, jars were placed in a water bath. For pre-hybridization, hybridization, antibody incubation and incubation during detection steps, the sections were covered with 200 ll of the appropriate solutions per slide and placed in a humid chamber. Tissue was rehydrated in a graded series of ethanol (95, 80 and 70%, 2 min each), brieﬂy rinsed in 29 sodium saline citrate buffer (SSC, Sigma-Aldrich, Munich, Germany), treated with 0.02 N HCl for 5 min, rinsed again in 29 SSC (2 9 5 min) and treated with freshly prepared acetylation solution containing 0.25% acetic anhydride and 0.2% HCl in 0.1 M triethanolamine buffer for 20 min, as previously described (Asan et al. 2005; Gutknecht et al. 2009). After rinsing in 29 SSC (2 9 5 min), sections were covered with pre-hybridization solution (50% deionized formamide, 49 SSC, 10% dextran sulfate, 19 Denhardt’s solution, 250 lg/ml salmon sperm DNA), and a piece of paraﬁlm was placed upon the section to evenly spread the solution. Incubation was carried out for 1 h at 57–58 C in a hybridization oven. Then, pre-hybridization solution was removed and replaced by one (for single FISH or CISH), two (for double FISH or FISH/CISH) or three cRNA probes of interest (for double FISH/CISH triple labeling) diluted in pre-hybridization solution at appropriate concentrations. Again, sections were covered with paraﬁlm and hybridiza- tion was carried out for 16–20 h at 57–58 C followed by post-hybridization washes: (1) 3 9 5 min in 29 SSC, (2) 50% formamide in 29 SSC at 58 C for 30 min, (3) 29 SSC (3 9 5 min), (4) RNase buffer (50 mM NaCl, 10 mM Tris, 1 mM EDTA, 40 lg/ml RNase A) at 37 C for 30 min and (5) RNase buffer without RNase A at 58 C for 30 min as described by Gutknecht et al. (2009). Afterwards, tissue was rinsed in washing buffer (100 mM Tris, 150 mM NaCl, pH 7.5) for 5 min and then treated with 0.5% blocking reagent (Perkin Elmer) diluted in washing buffer for 30–120 min. Detection protocols For single CISH, sections were subsequently incubated in AP-conjugated anti-Fluorescein (1:600, Roche) or anti- DIG Fab fragments (1:600, Roche) diluted in antibody incubation buffer (0.25% blocking reagent, 0.15% Triton X-100, 100 mM Tris, 150 mM NaCl) for 2.5 h, rinsed in washing buffer (2 9 5 min) and AP buffer (100 mM Tris, 100 mM NaCl, 50 mM magnesium chloride, pH 9.5) for 5 min and subjected to chromogenic detection with NBT/ 5-bromo-4-chloro-30-indolyphosphate (BCIP; Roche; stock 123