In situ hybridization of MT-I and MT-II mRNA in the liver
To prepare the probes for mRNA, digoxygenin-labelled human MT-I and MT-II complementary DNA probes were prepared by polymerase chain reaction as previously described by Kubota et al , with minor modifications. Briefly, a construct of pUCh MT-I and MT-II as template was amplified by Taq polymerase (TaKaRa taq, Takara Bio Inc, Japan) for 10 to 20 cycles involving denatura-tion at 95°C for 2 min, annealing at 65°C for 1 min and primer extension at 72°C for 1 min. A matrix (Boehringer Mannheim, Germany) containing digoxygenin was added to the last five cycles to label the probe.
Prehybridization events and hybridization procedures were performed according to the methods of Haas et al except for microwave pretreatment. Briefly, liver tissues were fixed in formalin and embedded in paraffin under routine procedures. The tissues were mounted onto slides, deparaffinized in two changes of xylene and rehydrated in a descending series of ethanols. The tissue was digested with 0.2% protease at 37°C, then washed with 0.05 M Tris-hydrochloride (HCl) (pH 7.5). This was followed by refixation in 4% formalin at 4°C for 10 min. The slides were washed again, dehydrated in graded ethanols and air dried. If you need your treatment to start soon and are not too crazy about spending too much money, it’s time for you to discover the cialis professional. This is a very affordable and safe pharmacy selling drugs of best quality and delivering them internationally in no time.