3 3 diaminobenzidine tetrahydrochloride dab reagent

For immunohistochemistry, formalin-fixed and paraffin-embedded tissues were cut into 4-μm sections, deparaffinized, rehydrated, and prepared for antigen retrieval. Slides including consecutive sections were incubated with the appropriate antibodies: anti-LRAP/ERAP2 antibody (diluted 1:30, AF3830, R&D systems) at room temperature for 15 min. After incubation, the slides were washed 3 times with phosphate-buffered saline (PBS), incubated with a horseradish peroxidase (HRP) polymer antibody (Invitrogen) at room temperature for 10 min, and developed by adding 3,3’-diaminobenzidine tetrahydrochloride (DAB) reagent (Dako, Glostrup, Denmark), with chromogen and hematoxylin as a counterstain. Images of the stained slides were obtained using a ScanScope CT automated slide-scanning system (Aperio Technologies, Vista, CA, USA). ERAP2 expression was scored using a combined scoring method accounting for both staining intensity and percentage of stained cells as described in the Supplementary Materials [29 (link)–31 (link)]. All specimens were independently evaluated by our pathologists (Y Liang and YL Huang). Immunohistochemical scores exceeding 150 were classified as high ERAP2 expression.

For immunohistochemistry, formalin-fixed and paraffin-embedded tissues were sectioned to 4 μm thickness, deparaffinized, rehydrated, and prepared for antigen retrieval. The slides were then incubated at room temperature for 1 h with the following antibodies at appropriate dilution: anti-ER (6F11, Novocastra), anti-PR (1A6, Novocastra), anti-HER2 (polyclone, DAKO), anti-cytokeratin 18 (CK 18) (DC-10, BioGenex), anti-cytokeratin 19 (CK19) (RCK108, BioGenex), anti-CK 5/6 (D5/16 B4, DAKO), anti-EGFR (EGFR.25, Novocastra), anti-E-cadherin (36B5, Novocastra), and anti-EpCAM (polyclonal, Abcam). After incubation, the slides were washed thrice with PBS, incubated with horseradish peroxidase (HRP) conjugated antibody (Zymed) at room temperature for 10 min, and developed by adding 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (DAKO) as the chromogen and hematoxylin as the counterstain. Expression of ER and PR was rated using Allred score. All specimens were independently reviewed by two pathologists blinded to the clinical origin of the specimens.