Diamine benzidine hydrochloride

Consecutive 4-μm sections were immunohistochemically stained using 0.2 μg/mL 4D3 and a previously described immunoperoxidase technique [39 (link)]. Secondary antibodies (Medical and Biological Laboratories, Nagoya, Japan) were used at a concentration of 0.2 μg/mL. Tissue sections were color-developed with diamine benzidine hydrochloride (DAKO, Glastrup, Denmark) and counterstained with Meyer's hematoxylin (Sigma). We counted immunopositive cells at the cytoplasmic membrane. Staining strength was scored from 0 to 3 (a score of 1 was used to describe the expression level in normal colonic epithelium). The staining index was calculated as the staining strength score multiplied by the staining area (%), and the resulting scores were defined as follows: none (index, 0), weak (index, 1–100), medium (index, 101–200), and high (index, 201–300). For a negative control, non-immunized rat IgG (Santa-Cruz Biotechnology, Santa-Cruz, CA, USA) was used as a primary antibody.
For immunostaining of spheres, spheres were treated with 4D3 and C225 antibodies labeled with HiLyte Fluor 555 and 647 (Dojindo, Kumamoto, Japan), respectively, for 4 h in accordance with the manufacturer's instructions. The spheres were observed by an all-in-one florescence microscope (Keyence Corp., Osaka, Japan).

Consecutive 4-mm sections were immunohistochemically stained using anti-biglycan mouse monoclonal antibody (0.2 e mon, clone 3E2, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-CD90 rabbit monoclonal antibody (0.2 d ant, clone EPR2959, Abcam plc., Cambridge, UK) or anti-CLDN4 antibody (0.2 μg/mL, clone 4D3), which was established in our laboratory [57 (link)], and a previously described immunoperoxidase technique [58 (link)]. Secondary antibodies for peroxidase-conjugated mouse IgG and alkaline phosphatase-conjugated rabbit IgG (Medical and Biological Laboratories, Nagoya, Japan) were used at a concentration of 0.2 μg/mL. Tissue sections were color-developed with diamine benzidine hydrochloride (DAKO, Glastrup, Denmark) for biglycan and with fast red (CosmoBio, Tokyo, Japan) for CD90. Slides were counterstained with Meyer’s hematoxylin (Sigma). Overexpression of biglycan was determined when the expression was stronger than that of biglycan in normal colon mucosa. For evaluation of CD90 immunostaining, number of positive cells was counted in 500 cells. For negative control, non-immunized rat IgG (Santa Cruz) was used as a primary antibody. Positive straining for biglycan was defined as stronger staining than that in normal colonic epithelium. We used placental tissue as a positive control.

Consecutive sections of 4 μm of BUC tissues were immunohistochemically stained using 0.2 µg/mL of primary antibodies using a previously described immunoperoxidase technique [39 (link)]. Secondary antibodies (peroxidase-conjugated antibodies, MBL, Nagoya, Japan) were used at a concentration of 0.2 µg/mL. Tissue sections were color-developed with diamine benzidine hydrochloride (DAKO, Glastrup, Denmark) and counterstained with Meyer’s hematoxylin (Sigma). The primary antibodies used are listed in Table 3.