3 30 diaminobenzidine solution

Tissue samples were formalin fixed, embedded in paraffin wax, and were cut into 4 μm. Paraffin sections were dewaxed in xylene and rehydrated through graded ethanol to water. Antigens were retrieved by boiling in 10 mM citriate buffer (pH 6.0) and endogenous peroxidase activity was quenched in methanol containing 3% hydrogen peroxide. The sections were incubated in PBS containing 3% BSA to block nonspecific binding, and were incubated for 30 min with primary antibodies including anti-GLP-1R (1:100), anti-p53 (1:50), anti-ER (1:50), and anti-PR (1:800). We tested normal endometrial tissue as a positive control, and negative control tissues were incubated without primary antibodies. The sections were subsequently incubated with secondary antibodies and Envision FLEX (DAKO). The antibody binding was visualized using a 3,30-diaminobenzidine solution (DAKO). After the sections were briefly counterstained with Mayer’s hematoxylin, the sections were dehydrated through a graded ethanol series and mounted.

Tissue samples were formalin fixed, embedded in paraffin wax, and cut into 4-μm sections. Paraffin sections were dewaxed in xylene and rehydrated through graded ethanol to water. Antigens were retrieved by boiling in 10 mM citrate buffer (pH 6.0) and endogenous peroxidase activity was quenched in methanol containing 3% hydrogen peroxide. The sections were incubated in phosphate buffered saline containing 3% bovine serum albumin to block nonspecific binding and incubated for 30 min with primary antibodies including anti-UCHL5 (1:100). We tested normal ovarian tissue as a positive control, and negative control tissues were incubated without primary antibodies. The sections were subsequently incubated with secondary antibodies and Envision FLEX (DAKO, Glostrup, Denmark). The antibody binding was visualized using a 3,30-diaminobenzidine solution (DAKO). After the sections were briefly counterstained with Mayer’s hematoxylin, the sections were dehydrated through a graded ethanol series and mounted.
UCHL5 cytoplasmic staining was scored according to its intensity as either negative (0), weakly positive (1), moderate positive (2), or strongly positive (3). UCHL5 immune expression was dichotomized into either low (score 0–1) or high (score 2–3), and the maximum score for each sample served for statistical analysis.