Jung cm 3000 cryostat microtome

The rats were sacrificed by decapitation 24 h after the last sucrose test. The brains were rapidly removed and frozen using a heptane–dry ice mixture. Coronal brain sections (12 μm) were cut using a Jung CM 3000 cryostat microtome (Leica, Germany). The slices were thaw mounted on gelatine-covered microscope slides, air dried, and stored at −20 °C until use. In order to compare obtained slices with The Rat Brain Atlas (Paxinos and Watson 1998 ), the cresyl violet staining was performed. Receptor autoradiography was carried out as described by Ferone et al. (1999 (link)). As radioligand, the Somatostatin-28, Tyr²⁵,[¹²⁵I]-Leu⁸, D-Trp²² (Perkin Elmer, Germany) in 0.1 nM concentration was used. Nonspecific binding was determined by 1 μM non labeled rat somatostatin-14 (SST14, Prospec, Israel). Data were analyzed using one-way analysis of variance (ANOVA). A post hoc Bonferroni test was performed to locate differences between group means. The criterion for statistically significant differences for all experimental groups was set at p < 0.05. Data are expressed as percent changes in specific binding compared with the control group (expressed as 100 %) using the GraphPad Prism 5.0.

The brains were isolated from decapitated animals, rapidly frozen using isopentane on dry ice, and stored at −80 °C until further stages. Afterwards, the brain Sects. (20 μm) containing the region of interest — the habenula — were cut using a Jung CM 3000 cryostat microtome (Leica, Wetzlar, Germany) according to Rat Brain Atlas [54 ]. The slices were then attached to PEN Membrane Glass Slides 2.0 µm (ThermoFisher Scientific, Waltham, MA, USA) and stained with Cresyl Violet from the LCM Staining Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Using laser capture microdissection (Leica, Wetzlar, Germany), the medial and lateral habenular nuclei were obtained.