Medicine of the Future in America

Interferon-alpha-induced changes in metallothionein expression: Methods (Part 3)

metallothionein geneAfter 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.

This entry was posted in Interferon-alpha and tagged Chronic hepatitis C, Interferon-alpha, Knodell histological activity index score, Metallothionein, PCNA labelling index.
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