Changes

Reference: Swart, I., J.M. Overton, and T.A. Houpt. Hypothalamic NPY, AGRP and POMC mRNA responses to leptin and refeeding in mice. Am. J. Physiol. 283 (2002) R1020-R1026. PMID 12376393.

Reference: Swart, I., J.M. Overton, and T.A. Houpt. Hypothalamic NPY, AGRP and POMC mRNA responses to leptin and refeeding in mice. Am. J. Physiol. 283 (2002) R1020-R1026. PMID 12376393.

Coronal 40 micron sections were cut on a sliding freezing microtome through the rostral caudal extent of the hypothalamus. Alternate sections were placed into ice-cold 2x SSC (SSC = 0.15M NaCl/0.015M Na Citrate). Free floating tissue sections were prehybridized in glass vials in 1ml of 60% formamide, 0.02 M Tris pH7.4, 1mM EDTA, 10% dextran sulfate, 0.8% Ficoll, 0.8% PVP, 0.8% BSA, 2x SSC, 0.1M dithiothreitol, and 1.6 mg/mL herring sperm DNA for 2h at 37°C. After 2h prehybridization, radiolabeled probe was added (~1.0 x 107 cpm/vial) and incubated for 16-20h at 37°C. Hybridization was performed with either tail labeled prepro NPY (ppNPY) antisense oligonucleotide (bases 59-88), agouti-related protein (AGRP) antisense oligonucleotide (bases 1-45) or proopiomelanocortin (POMC) antisense oligonucleotide (bases 482-517). The oligonucleotides were tail-labeled to approximately equal specific activities (~10^7 cpm/100ng) with 35S-dATP by terminal transferase reaction (Roche),  Sections were then sequentially rinsed in 2x SSC, 1x SSC and 0.5x SSC for 10 minutes at 37°C. The tissue sections were mounted from 0.05M sodium phosphate onto gelatin- coated slides, air dried, and exposed to autoradiographic film (β-max, Amersham) for 2-3 days. Tissue sections from different groups were hybridized in parallel and exposed to film together to ensure that in situ hybridization was carried out on representative members of each experimental group at the same time under identical conditions, allowing direct comparison of mRNA expression.

Coronal 40 micron sections were cut on a sliding freezing microtome through the rostral caudal extent of the hypothalamus. Alternate sections were placed into ice-cold 2x SSC (SSC = 0.15M NaCl/0.015M Na Citrate). Free floating tissue sections were prehybridized in glass vials in 1ml of 60% formamide, 0.02 M Tris pH7.4, 1mM EDTA, 10% dextran sulfate, 0.8% Ficoll, 0.8% PVP, 0.8% BSA, 2x SSC, 0.1M dithiothreitol, and 1.6 mg/mL herring sperm DNA for 2h at 37°C. After 2h prehybridization, radiolabeled probe was added (~1.0 x 107 cpm/vial) and incubated for 16-20h at 37°C. Hybridization was performed with either tail labeled prepro NPY (ppNPY) antisense oligonucleotide (bases 59-88), agouti-related protein (AGRP) antisense oligonucleotide (bases 1-45) or proopiomelanocortin (POMC) antisense oligonucleotide (bases 482-517). The oligonucleotides were tail-labeled to approximately equal specific activities (~10^7 cpm/100ng) with 35S-dATP by terminal transferase reaction (Roche),  Sections were then sequentially rinsed in 2x SSC, 1x SSC and 0.5x SSC for 10 minutes at 37°C. The tissue sections were mounted from 0.05M sodium phosphate onto gelatin- coated slides, air dried, and exposed to autoradiographic film (β-max, Amersham) for 2-3 days. Tissue sections from different groups were hybridized in parallel and exposed to film together to ensure that in situ hybridization was carried out on representative members of each experimental group at the same time under identical conditions, allowing direct comparison of mRNA expression.