For analysis, the membranes were exposed for 15 and 24 h to a phosphor screen and detected using a Bio-imaging Analyzer BAS2000 (Fuji Photo Film Co. Ltd Japan.
Description
Hypothalamus of rats befrm (2d) heat acclimation and hypohydration (10% loss of b. wgt, water deprivation). RNA extraction was accomplished using Tri Reagent (Molecular Research Center Inc. Ohio, USA). The probes were labeled for 1 h by the reverse transcription of total RNA (3 µg) at 42°C in a primer mix (Clontech) containing [32P]dATP (Amersham Biosciences, Buckinghamshire, UK). The reaction was terminated by adding 0.1M EDTA and 1 mg/ml glycogen (Sigma).The unincorporated 32P-labeled nucleotides were removed by Nucleo-Spin extraction columns (Clontech).
Data processing
We used Atlas Image 2.01 software (Clontech) to grid the phosphor image, to record the pixel density of each spot, and to perform background subtraction. The background-subtracted data were then further analyzed.
