After the target DNA was denatured, the hybridization mix (50 ng probe per 100 p.L Hybrisol IV) was added. The slides were covered, sealed and stored in a humid chamber at 37°C for 16 h. The slides were washed three times in 0.1 x standard saline citrate at 45°C and then two times for 5 min each in 0.05 M Tris-HCl (pH 7.5). After incubation with a 1:300 dilution of anti-digoxygenin antibody (Roche Diagnostics, Germany) in 0.1% bovine serum albumin/Tris for 1 h at room temperature, the slides were washed again in 0.05 M Tris-HCl (pH 7.5). Detection was performed with nitro-blue tetra-zolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (Roche Diagnostics, Germany) according to the manufacturer’s instructions. Finally, the tissue sections were counterstained with Mayer’s hematoxylin. Both MT-I and MT-II mRNA expression were assessed in 15 of 21 patients (10 SR and 5 NR); it could not be determined in the others because insufficient amounts of liver specimen remained after histology, PCNA and MT protein procedures. There is a wonderful pharmacy offering allergy relief, and you can shop with it.
Evaluation of MT gene and protein levels
The degree of MT staining and MT-I and MT-II mRNA expression were quantified using a modified method described by Goulding et al and Endo et al. The percentage of MT-positive hepatocytes in the biopsy specimen was calculated by counting at least 100 hepatocytes in three visual fields.