Ab252224

A portion of the formalin-fixed paraffin-embedded liver tissue samples, obtained at enrolment in the study, was used for SIX1′s the immunohistochemical evaluation. After deparaffinization and rehydration, antigen unmasking was performed with 1 mM EDTA buffer, pH 8, at 98°C for 15 mi. Then, these sections were incubated in methanol 5% and H₂O₂ 1% for five minutes for blocking endogenous peroxidases, whereas nonspecific sites were blocked using a blocking solution reagent with bovine serum albumin 3% for 30 min at room temperature. Sections were incubated with mouse anti-SIX1 (AB252224; Abcam) primary antibody at a working dilution of 1:60. Thereafter, sections were incubated with prediluted OmniMap anti-mouse horseradish peroxidase-conjugated secondary antibody (Ventana Medical Systems from Tucson, AZ), for 20 min in a humidity chamber as well as with detection kit reagents (ultra-view universal horseradish peroxidase multimer and diaminobenzidine [DAB] chromogen, Ventana Medical Systems) in compliance with the manufacturer's instructions. Subsequently, these sections were counterstained with haematoxylin, dehydrated, and permanently mounted for microscopic examination). To obtain the intensity value of the DAB signal, images of stained liver tissue were processed with ImageJ software (http://rsbweb.nih.gov/.