Ihc tektm epitope retrieval solution

The liver samples were fixed in neutral-buffered formalin and embedded in paraffin blocks according to standard procedures. Paraffin-embedded tissue sections were incubated in xylene for 15 min. After rehydration in graded ethanol, antigen retrieval was performed in IHC-Tek^TM Epitope Retrieval Solution (IHC World, Ellicott City, MA, USA) by heating in a retrieval steamer for 40 min. Then, the sections were allowed to cool for 20 min. Subsequently, the sections were incubated with 3% H₂O₂ at room temperature for 30 min, blocked for 5 min, and then incubated with anti-8-oxo-dG (8-oxo-2′-deoxyguanosine) antibodies (Trevigen, Gaithersburg, MD, USA) overnight. The next day, the sections were incubated with the goat anti-mouse IgG HRP-conjugated secondary antibody (Merck Millipore, Temecula, CA, USA) for 30 min, washed with Dulbecco’s phosphate-buffered saline containing Tween-20, and then stained with 3,3′-diaminobenzidine. Hematoxylin and eosin and Sirius Red staining were processed to confirm the pathological analysis of liver disease by the KPNT (Korea Pathology Technical Center, Cheongju, Korea). Histological scoring (Supplementary MaterialsTable S1) was performed as previously described [56 (link)].

Formalin-fixed paraffin-embedded (FFPE) tumor tissue sections were deparaffinized in xylene and then subjected to a gradient of alcohol, followed by retrieval in IHC-TekTM Epitope Retrieval Steamer with IHC-TekTM Epitope Retrieval Solution (IHC World) following the instruction of the manufacturer. To block endogenous peroxidase activity, the sections were incubated in 3% H₂O₂ for 10 min, followed by incubation in blocking buffer containing 2% horse serum, 1% BSA, 0.1% Triton X-100, 0.05% Tween 20 in PBS at room temperature for 30 min. Then sections were incubated with anti-CXCR4 antibody (1:400, Abcam) at 4 °C overnight. After washing, sections were incubated with goat anti-rabbit IgG-HRP (1:500, Abcam) for 1 h and visualized with DAB. The slides were analyzed with Keyence Microscope BZ-X810.