For immunohistochemistry [14 (link)], paraffin sections were treated with hydrogen peroxide to inactivate endogenous peroxidases. Antigen retrieval was performed in a microwave in 10 mM citrate buffer at pH 6.0. Sections were fixed with paraformaldehyde followed by permeabilization and blocking. Sections were then incubated in anti-URG11 (Santa Cruz) antibody overnight at 4°C, and a secondary antibody was used to detect protein expression. Immunostaining was analysed with the Super Sensitive Non-Biotin Polymer HRP Detection System according to the manufacturer's instructions (BioGenex, San Ramon, Canada).
For analysis, randomly selected five fields of the staining were captured; the grade values were determined by the percentage of positive stained cells and the strength of URG11 staining [15 (link)]. Briefly, the ratio of positive cells scored 0 for staining of ≤1%, 1 for staining of 2 to 25%, 2 for staining of 26 to 50%, 3 for staining of 51 to 75%, and 4 for staining >75%. Staining intensity was graded as follows: negative: 0; weak signal: 1; middle: 2; and strong: 3. Then the total score resulting from the multiple value of positive percentage score and strength score is as follows: 0-1: grades negative (-); 2-4: weak positive (+); 5-8: middle (++); and 9-12: strong (+++).
For analysis, randomly selected five fields of the staining were captured; the grade values were determined by the percentage of positive stained cells and the strength of URG11 staining [15 (link)]. Briefly, the ratio of positive cells scored 0 for staining of ≤1%, 1 for staining of 2 to 25%, 2 for staining of 26 to 50%, 3 for staining of 51 to 75%, and 4 for staining >75%. Staining intensity was graded as follows: negative: 0; weak signal: 1; middle: 2; and strong: 3. Then the total score resulting from the multiple value of positive percentage score and strength score is as follows: 0-1: grades negative (-); 2-4: weak positive (+); 5-8: middle (++); and 9-12: strong (+++).
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