Immuno-histochemical studies

For the immunohistochemistry detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG), Alpha-smooth muscle actin (-SMA), and BAX, paraffin blocks of formalin-fixed SMG tissue specimens were used using the streptavidin–biotin immunoperoxidase technique (Dako- Cytomation, Glostrup, Denmark). Sections of 3–5 mm thickness were cut from formalin-fixed, paraffin-embedded blocks, placed on positively charged slides, deparaffinised in xylene, and rehydrated in graded alcohol. A citrate buffer (pH 6.0) was used to boil the sections for 20 min. They were then washed in PBS (pH 7.3). Sections were treated with 3% hydrogen peroxide to block the endogenous peroxidase activity. Thereafter, the sections were incubated with the primary antibody. These antibodies included Glypican-3 (GPC-3): mouse monoclonal antibody, dilution 1:100; (CM396, A, B, Clone IG12, Biocare Medical LLC, Concord, USA); 8-Hydroxydeoxyguanosine(8-OHdG): monoclonal antibody, dilution 1:100 (Clone 15A3, SC-66036, Santa Cruz, California, USA), mouse monoclonal antibody to α-SMA 1/500 dilution (Clone 1A4, ab7817, Abcam) in a humid chamber overnight, then washed three times with PBS (Ramos-Vara et al., 2008). For BAX sections, they were first incubated with 1% pre-immune rabbit serum to decrease nonspecific staining and then with a monoclonal antibody against BAX protein (1:200 dilution; Dako, Carpinteria, California, USA)³⁹.

Incubation with a secondary antibody and product visualisation were performed (Dako-Cytomation). For 8-OHDG, diaminobenzidine substrate (Research Genetics, Huntsville, Alabama, USA) was the chromogen, and for α-SMA 3,3'-diaminobenzidine (DAB) hydrogen peroxide was used as a chromogen to stain α-SMA bounded structures and localise the site of immune reactions. For BAX, 3-amino-9-ethyl carbazole (AEC) diaminobenzidine substrates were used as chromoge; finally, immune-stained sections were counterstained using Mayer's haematoxylin.