Peptidases and Neuropeptide Processing, Volume 23: Volume 23 by A. Ian Smith

By A. Ian Smith

The volumes during this sequence comprise modern concepts major to a selected department of neuroscience. they're a useful reduction to the coed in addition to the skilled researcher not just in constructing protocols in neuroscience yet in disciplines the place study is changing into heavily concerning neuroscience. every one quantity of equipment in Neurosciences comprises an index, and every bankruptcy comprises references. Dr. Conn grew to become Editor-in-Chief of the sequence starting with quantity 15, so each one next quantity might be guest-edited via a professional in that express box. This additional strengthens the intensity of insurance in tools in Neurosciences for college kids and researchers alike.

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Riboprobes are diluted in the hybridization mix to a final concentration of 5 x 106 to 5 x 107 dpm/ml hybridization solution and dithiothreitol (DTT) is added to the final concentration of 10 mM. The addition of DTT is important when using 35S-labeled probes to prevent the formation of covalent disulfide bonds, which causes irreversible nonspecific binding of the probe to sulfated groups in the tissue. This solution can be stored at -80~ for several weeks. We have found that the degradation of the radiolabeled probes in the hybridization solution is greatly reduced.

25% (v/v) acetic anhydride (A6404; Sigma) and incubated under vigorous stirring for 10 min at room temperature. 015 M sodium citrate). Dehydration and Delipidation Slides are dehydrated through a graded series of ethanol (50, 70, 95, and 100%), immersed in chloroform for 5 min, rinsed in 100% ethanol, 95% ethanol and then air dried. We typically apply the probe diluted in hybridization buffer immediately after the prehybridization. However, tissue sections can be stored for several days at room temperature and in a desiccated form for several months at -20~ prior to hybridization.

Total RNA from rat anterior pituitary was reverse transcribed. For PCR amplification oligonucleotides comprising mouse PC1 (mPC1)-specific sequences were used" nucleotides 715-737 (forward primer: 5' GGT GAA ATT GCC ATG CAA GCA AAT 3') and mPC1 nucleotides 1206-1186 (reverse primer: 5' GGTTCTCTGTG CAGTCATTGTG 3'). The amplified 492-bp segment subcloned into the transcription vector Bluescript KS + (Stratagene, La Jolla, CA) was completely sequenced and showed a 97% identity to the corresponding mPC 1 sequence.

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