The mixture of Cy3- and Cy5-labeled probes was applied at 75°C onto arrays, and hybridized for 3 h at 65°C, 3 h at 55°C and 12 h at 50°C in the dark, using an automatic hybridizer, GeneMachines HybStation (Genomic Solutions).
Labelled targets (1.5 μg cRNA per target) were hybridised overnight on prehybridised murine oligonucleotide microarrays (65-mer with 5'-hexylaminolinker, Sigma-Genosys mouse 7.5K oligonucleotide library, printed in duplicate) using an automatic hybridisation station (Gene Tac Genomic Solutions).
The temperature of the reacting solution was controlled, using an automatic circulation thermostat.
The matrix solution was then deposited using an automatic vibration vaporization system (ImagePrep, Bruker Daltonics, Bremen, Germany) to cover the whole surface of the tissue section.
The water was maintained at 25 ± 1°C, made opaque by the addition of 200 ml of latex solution, and changed daily using an automatic filling and draining system.
Aliquots of 50 µl of this solution were spread on Mueller Hinton agar plates in duplicate using an automatic spiral plater (model WASP 2, Don Whitley, Shipley, United Kingdom).
The perfusion solution was delivered at a constant volume of 10 ml/min using an automatic pump.
After 20 min incubation in room temperature, the stop solution (0.1 N sulfuric acid) was added and the optical density was determined at 450 nm using an automatic microplate reader (Bio-Rad, USA).
Experimental pH was adjusted using an automatic CO2 injection system.
[ 17, 18] using an automatic analyzer.
