Blocking sniper

We performed immunohistochemical staining using the previously described method [43 (link)]. Briefly, slides were deparaffinized for 15 min at 50–60 °C followed by treatment with xylene twice for 5 min. The tissue sections were rehydrated by sequential treatment with 100%, 95%, and 80% alcohol. Peroxidase quenching was carried out by incubation with 3% hydrogen peroxide and 100% methanol for 5 min. The slides were placed in a plastic coplin jar with Reveal Decloaker RTU (Biocare Medical, Pacheco, CA, USA) for 25 min at 95 °C in a steamer for heated antigen retrieval. Following this step, the slides were allowed to cool at room temperature for 20 min. The tissue sections were rinsed in deionized, distilled water and marked using an ImmEdge hydrophobic barrier pen. The slides were incubated with a blocking sniper (Biocare Medical, Pacheco, CA, USA) for 20 min. Immunohistochemical staining of β-catenin, p-ser9-GSK-3β and glutamine synthetase (GS) in HCC tissues and normal liver tissues were examined.

IHC assays were performed as we described previously [47 (link)–49 (link)]. In brief, the tumors were formalin-fixed and paraffin-embedded. Five-micron-thick sections were deparaffinized in xylene and rehydrated with a series of graded alcohols. The slides were then treated with 3% hydrogen peroxide in methanol for 15 min. To exhaust endogenous peroxidase activity, the antigens were retrieved in 0.01 M sodium cirate buffer (pH 6.0) using a microwave oven. The slides were blocked with a blocking sniper (Biocare Medical, Pacheco, CA), and then incubated with a primary Ab described in the figure legends at 4°C overnight. After washing with Tris Buffer Saline (pH 8.0), the slides were incubated with a MACH 1 HRP Polymer detection kit (Biocare Medical) according to the manufacturer’s instructions. The staining colors were developed with a DAB Chromogen Kit (Biocare Medical). Finally, all sections were counterstained in Mayer’s hematoxylin, nuclei blued in 1% ammonium hydroxide (v/v), dehydrated, and then mounted with permanent aqueous mounting medium (Bio-Rad). Two individuals independently evaluated the IHC slides.