Diaminobenzidine tetrachloride

For immunohistochemical analyses of GBM, paraffin-embedded samples were sliced and mounted on microscopic slides. Rabbit monoclonal anti-SNAI2 and anti-E-cadherin antibodies (1:200 dilutions, Cell Signaling Technology, USA) were used as the primary antibodies. Heat-induced epitope was formed with a microwave in 10 mmol/l citric acid buffer at pH 7.2. The samples were incubated with the antibody overnight in the same buffer followed by incubation with biotinylated secondary antibody (1:500 dilutions, Santa Cruz Biotechnology, USA). The bound antibodies were visualized by the avidin biotinylated peroxidase complex methods and diaminobenzidine tetrachloride (Santa Cruz Biotechnology).

Formalin-fixed paraffin-embedded tissue sections were deparaffinized inxylene on microscopic slides. Antigen retrieval was performed by microwaving the sections in 10 mM citric acid buffer (pH 7.2). The primary antibodies used in this study were: anti-human IDH1-R132H monoclonal antibody (1:100, IBL Co., Ltd, Gumma, Japan) and anti-MGMT monoclonal antibody MT3.1 (1:200, Chemicon, Inc., Temecula, CA). The samples were incubated with the primary antibody overnight, followed by incubation with a biotinylated secondary antibody (1:500, Dako, Tokyo, Japan). The bound antibodies were visualized using the avidin biotin peroxidase complex method and diaminobenzidine tetrachloride (Santa Cruz Biotechnology, Inc.). To evaluate IDH1 staining, strong cytoplasmic staining in any number of cells was scored as positive. For MGMT scoring, the positive cells in a 200 × field (minimum of 1,000 nuclei) were counted, and the labeling index was expressed as a percentage of the labeled tumor cells. MGMT protein expression ≥10% was considered positive.

Paraffin-embedded SCLC samples were sectioned and mounted on microscopic slides. Polyclonal anti-Kir2.1 and anti-MRP1/ABCC1 antibodies (Alomone Labs Ltd., Israel) was used as the primary antibodies. Antigen retrieval was performed by microwaving in 10 mmol/L citric acid buffer at pH 7.2. The samples were incubated first with the primary antibodies overnight at 4°C, then with the secondary antibodies for 2 h at room temperature in the same buffer and finally with abiotinylated secondary antibody (DAKO, Tokyo, Japan). The bound antibodies were visualized using the avidinbiotinylated peroxidase complex and diaminobenzidine tetrachloride methods (Santa Cruz Biotechnology).
The IHC-stained samples were independently evaluated for Kir2.1 and ABCC1 expressions by two pathologists blinded to the clinical parameters. The staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) or 3 (strong). The extent of staining was scored as 0 (0-5%), 1 (6-25%), 2 (26-50%) or 3 (51-100%), according to the percentages of the positive stained area in relation to the entire carcinoma-involved area or the entire normal sample area. The sum of the intensity and extent scores was used as the final staining score (0–6). Optimal cut off values were identified; a final staining score ≤ 1 indicated negative expression and a final staining score >1 indicated positive expression.