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The resected tumors tissues were cut into 5-μm sections and fixed in paraformaldehyde. IHC stain was employed to detect the expression of Sulfatase-1 and Mesothelin. The tissues were fixed in 4% buffered formalin for 15 min at 4°C and rinsed in TBS. paraffin sections were dewaxed in xylene and rehydrated in a series of ethanol solutions after which antigen retrieval was done with Tris buffer at pH 9.0 in a microwave. Endogenous peroxidase activity was blocked by 20 min pre-incubation with 3% H₂O₂ and then incubated with the blocking solution (Horse serum) for 30 min at room temperature. The incubation with primary antibodies (goat monoclonal anti- Sulfatase 1 antibody, Abcam, China, 1-3μg/ml, and anti- Mesothelin antibody, Abcam, China, 1μg/ml) was carried out overnight at 4°C. it was then Incubated with the secondary antibody for 1hr and color was developed with diaminobenzidine (DAB) (Zhongshan Biotechnology, China). The positive reaction manifested as a brown stain. The section was counterstained in Mayor's haematoxylin. Dehydration process started from 80%, 95% and 100% of ethanol each for 2 min and then washed with xylene 2times for 2 min each. Mounting and cover slipping were done and we proceeded to slide reading. Other slides were stained by routine Haematoxylin and Eosin staining method and the rate of lymph node metastasis was determined.

The tissue slide was baked in a dry oven at 60°C for 2 h to remove the coated paraffin. The samples were demineralized with an equal parts mixture of 20% sodium citrate and 45% formic acid. The slide was immersed twice in xylene for 3 min, hydrated with 100%, 95%, 70%, and 50% ethanol, and rinsed with cold tap water for 5 min. After dewaxing and blocking endogenous peroxidases, the sections were treated at 100°C in EDTA (1 mM, pH 8.0) for antigen retrieval and then incubated with Eag1 polyclonal antibody (ab86204, Abcam, Cambridge, MA, 1 : 300) overnight at 4°C. The slides were washed with PBS and incubated with horseradish peroxidase conjugated goat anti-rabbit IgG at room temperature for 1 h. Diaminobenzidine (DAB, Zhongshan Biotechnology, Beijing, China) was used to visualize the tissue slide and the sections were counterstained with haematoxylin. Samples were defined as positive when more than 10% of the cells stained positive with Eag1 antibody. Eag1 staining was classified as 0 (less than 10% of the tumor cells show staining), 1+ (faint staining in more than 10% of the cells), 2+ (moderate staining in more than 10% tumor cells), and 3+ (strong staining in more than 10% of the cells) and the immunohistochemical score was evaluated as negative (0), positive (1), and strongly positive (2 and 3) as described previously [17 ].