Two step envision kit

TMA sections were processed for immunohistochemistry (IHC) after depara nization and rehydration routine procedures. Antibody antigen retrieval was performed using tissue submersion in citrate buffer (10 mM, pH 6.0) with heating. Sections were then incubated with an anti-PRR11 antibody (HPA023923, Sigma-Aldrich; Saint Louis, MO, US), anti-ki-67 (cat. 27309-1-AP, proteintech; Wuhan, China), anti-Ncadherin (cat. 610920, BD Biosciences; San Jose, CA, US) and anti-early growth response protein 1 [EGR1] (ab54966, Abcam; Cambridge, UK) at 4℃ overnight. Subsequently, a two-step Envision kit (Agilent Technologies; Santa Clara, CA, US) was used to visualize positive staining. PRR11, N-cadherin, and EGR1 protein expression was evaluated by two researchers who were blind to this study using an Olympus CX31 microscope (Olympus Co; Tokyo, Japan). The antibodies were diluted with 1:100 goat serum for IHC analysis. Positivity was calculated by the semiquantitative scoring system described previously [15] . Generally, IHC staining intensity was assigned as: negative, 0; weak, 1; moderate, 2; and intense; 3. The percentage of IHC positive cells was scored as 0 to 1 (0% to100%). Theoretically, a weighted score ranging from 0 (0% of cells staining) to 3 (100% of the cells staining at 3+ intensity) was generated for each tissue core. An IHC score bigger than (>) 0 was considered positive.