Biotin labeled secondary antibody

Immunohistochemical staining was performed on 4 μm tumor tissue sections. The section was deparaffinized, rehydrated, and autoclaved in a 0.01 M citrate buffer solution (pH 6.0). Section was later incubated overnight with anti-Ki-67 antibody (1:500; Cell Signaling Technology), followed by incubation with biotin-labeled secondary antibody (1:5000; Thermo Scientific, Waltham, USA) at room temperature for 20 min. The section was detected using 3,3′-diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, and visualized under a light microscope (Olympus, Tokyo, Japan).

TMA sections were then used for the immunohistochemical staining as described previously.24 (link) Briefly, TMA sections were deparaffinized with xylene and then dehydrated through descending grades of ethanol to deionized water according to standard procedures. After antigen retrieval with the appropriate buffer (0.01 M citrate buffer, pH 6.0, high to boiling) for 3 minutes, the sections were cooled at room temperature. Endogenous peroxidase was blocked with 3% (v/v) aqueous hydrogen peroxide for 10 minutes, followed by incubation with normal serum for 20 minutes to reduce nonspecific binding. Then, the sections were incubated with a primary antibody against SP (1:100, SC9758; Santa Cruz Biotechnology Inc., Dallas, TX, USA) or an antibody against NK1R (1:250, SC14116; Santa Cruz Biotechnology Inc) overnight at 4°C. Negative controls were included, and steps were carried out with PBS. Subsequently, TMA sections were incubated with biotin-labeled secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 20 minutes at room temperature, followed by incubation with streptavidin-biotinylated horseradish peroxidase-conjugated antibody (Thermo Fisher Scientific) for another 20 minutes. Finally, the sections were stained with 3,3-diaminobenzidine and lightly counterstained with Mayer’s hematoxylin, dehydrated, and mounted with resinous mounting medium.