Based on these sequences, we designed specific primers and probes for real-time PCR analysis and in situ hybridization.Fluorescence in situ hybridizationTRH and D2 mRNA expression was analyzed in two healthy vs. two prolonged ill animals. The TRH and D2 in situ probes were generated in our laboratory. The TRH probe was complementary to 1 to 203 bp of the rabbit proTRH mRNA sequence (“type”:”entrez-nucleotide”,”attrs”:”text”:”EU489480″,”term_id”:”169743248″,”term_text”:”EU489480″EU489480), and the D2 probe was complementary to 3230 to 608 bp of the rabbit type II deiodinase gene (“type”:”entrez-nucleotide”,”attrs”:”text”:”EF370408″,”term_id”:”126572616″,”term_text”:”EF370408″EF370408). The fluorescent in situ hybridization protocol was adapted from a standard in situ hybridization protocol of the TSA? Biotin System (New England Nuclear, Boston, MA). The antisense RNA probes were digoxin/digoxigenin labeled, diluted in hybridization buffer and hybridized on 10 ��m tissue cryosections overnight in a humidified stove at 62��C. After washing, the sections were incubated with an anti-digoxin/digoxigenin horseradish peroxidase-labeled antibody. The probe was amplified using Tyramide Amplification Reagent (TSA? Biotin System, NEN, Boston, MA), and visualized with streptavidine conjugated to Cy3. Following washes, the sections were mounted in Fluorescent Mounting Medium (DAKO, Glostrup, Denmark) with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, Bornem, Belgium) to counter stain cell nuclei. We analyzed sense-probes as negative controls. As it is not possible to quantitate data obtained from fluorescent in situ hybridization with tyramide amplification, we attempted to do isotopic in situ hybridizations. This resulted in high background and low signal to noise ratio and results could not be used for analysis.RNA isolation and real-time PCRGene expression analysis was performed on eight healthy rabbits vs. six prolonged ill animals. RNA was isolated from the total hypothalamic block using the RNeasy midi RNA isolation kit (Qiagen, Venlo, The Netherlands) and quantified by Nanodrop spectrophotometer (ND-1000, Nanodrop Technologies, Wilmington, DE, USA). Samples were treated with DNAse to remove all contaminating genomic DNA. A 1 ��g sample of total RNA was reverse-transcribed using random hexamers. All samples were reverse transcribed simultaneously. Reactions lacking reverse transcriptase were also run as a control for genomic DNA contamination.