Dako universal lsab kit

Immunolocalization for hepatic tumor necrosis factor-α (TNF-α) was applied on sections of 5-μm thickness and stained with the streptavidin-biotin-peroxidase staining technique [20 , 42 (link), 43 (link)] using an antisera containing 1^ry antibodies for rat anti-TNF-α (Santa Cruz, CA, USA), biotinylated 2^ry antibodies and streptavidin horseradish peroxidase (Dako-K0690; Dako Universal LSAB Kit), and diaminobenzidine tetrahydrochloride (DAB) substrate kit (Sigma-D5905; Sigma–Aldrich Company Ltd., Gillingham, UK) for 10 min for immunolabelling. All prepared sections were treated under the same conditions with the same antibody concentrations simultaneously, so the immunostaining among the different groups was comparable.

The sections were stained following the streptavidin-biotin-peroxidase staining method [23 (link)]. Paraffin tissue sections (4–6 μm) were deparaffinized in xylene and then rehydrated in ethanol. Endogenous peroxidase and nonspecific binding sites for antibodies were suppressed by treating the sections for 10 min with hydrogen peroxide (0.3%) and for 20 min with 5% normal bovine serum (1 : 5 diluted tris buffer saline [TRIS]) at ambient temperature, respectively. Obtained sections were rinsed with PBS, and then, 10% normal goat serum was applied for 30 min to minimize nonspecific binding. Obtained sections were then incubated in anti MMP13 primary antibody (cat no: GB11247; Servicebio, China) for 1 h, followed by incubation in streptavidin horseradish peroxidase (Dako-K0690) and biotinylated secondary antibody (Dako Universal LSAB Kit) for 15 min and then incubated in 3-diaminobenzidine tetrahydrochloride (Sigma-D5905; Sigma-Aldrich Company Ltd., Gillingham, UK) substrate kit for 10 min to achieve immunolabelling. Afterward, the nuclei were stained using Harry's hematoxylin stain, dehydrated ethanol, cleared in xylene, and then mounted in DPX. Antibody binding was observed with high-power light microscopy.