Hilyte fluor 555

Mitsuba-1 and MytiLec-1 (100 μg/L), after dialysis against 100 mM NaHCO₃ in saline, were labeled with HiLyte Fluor 555 (Dojindo Molecular Technologies Inc., Kumamoto, Japan) according to the manufacturer’s instructions. Labelled lectin was incubated with Raji cells (5 × 10⁵, in 100 μL culture medium) for 30 min at room temperature. Cells were then washed three times with culture medium, and fluorescence was observed with a BZ-X700 microscope (Keyence Corporation, Osaka, Japan) using 555 nm (excitation) and 570 nm (emission).

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).