Envision rabbit hrp kit

For the immunohistochemical examination of the OSCC tissue samples, 5 μm thick sections were deparaffinized and rehydrated. For antigen retrieval, tissue samples were incubated in citrate buffer at pH 6 in a microwave oven for 3 × 5 min at 750 W, and then, were cooled to room temperature (RT). Tissue endogenous peroxidase activity was inhibited by incubation of the sections in 3% hydrogen peroxide (H₂O₂) for 20 min. Samples were incubated in normal goat serum for 20 min in order to prevent nonspecific binding of the secondary antibody. Sections were then first incubated at a 1:1000 dilution with rabbit polyclonal anti-TRPA1 antibody (TRPA1: Novus 40763) for 1 h at RT. After several washing steps, sections were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (EnVision Rabbit HRP Kit, Dako-Cytomation, Carpinteria, CA, USA) for 30 min. Sections from normal mucosa incubated without primary served as negative controls. The TRPA1-like immunopositivity of the OSCC sections was examined under bright-field microscope (Olympus, BX51). Furthermore, we tested this antibody on the stable human TRPA1 expressing CHO cell line (dilution: 1:1000) with Cy5-conjugated anti-rabbit secondary antibody (GYÁRTÓ). The immunopositivity was examined with a Nikon Eclipse XXX fluorescent microscope.