Super sensitivetm link label ihc detection system

Immunohistochemistry (IHC) of liver biopsy tissue sections was performed as described previously^{50 (link)}. Briefly, liver sections were deparaffinized in xylene and rehydrated through a series of graded ethanol. Antigen retrieval was performed by placing the tissue sections in a Tris-EDTA buffer containing 10 mM Tris-HCl (pH 9.0), 1 mM EDTA and 0.05% Tween 20, and heated to above 65 °C in a microwave for 10 min. The endogenous peroxidase was quenched by using 3% hydrogen peroxide for 10 min. Non-fat dried milk (5%) was applied to the sections at room temperature for 1 h to block the non-specific antibody binding sites. After incubating with the indicated antibodies overnight at 4 °C, the tissue sections were washed extensively using 1X PBS. The antigen-antibody complexes were then detected by using the Super Sensitive^TM Link-Label IHC Detection System (BioGenex, Fremont, CA) and the cell nucleus was counter-stained by hematoxylin for 30 sec. After another extensive wash, the sections were dehydrated through a graded ethanol series ending in water, mounted and observed by microscopy (Olympus BX50).

Sections of tissues (4 μm thick) from microarrays were deparaffinized and rehydrated before staining. Tissue antigens were retrieved by autoclaving in 10 mM (pH 6) citrate buffer for 10 min. Sections were cooled on ice for 30 min before treatment with 3% H₂O₂. Samples were permeabilized with 0.2% Triton X-100 (Sigma) in DPBS and reacted with a diluted primary STMN1 antibody (1:200, 13655S, Cell Signaling). Signals were amplified and detected with a Super Sensitive^TM Link-Label IHC Detection System (BioGenex, Fremont, CA, USA) according to the instructions and counterstained with hematoxylin QS (Vector, Burlingame, CA, USA) for 20 s. The H-scores represent the percentage of STMN1 immunoreactivity-positive regions multiplied by the STMN1 staining intensity. Images were acquired with a BX43 light microscope equipped with a DP22 CCD camera (Olympus).