Immunocytochemical procedures were performed as described previously [17 (link)]. Briefly, cells were collected by pipetting with PBS and plated on coverslips coated with poly-L-lysine (Sigma-Aldrich) and fixed. After incubation in the digestion buffer with or without protease-free Chase ABC (1 U/mL, Seikagaku Corporation) at 37°C for 2 h, cells were incubated in blocking solution containing 2% bovine serum albumin, 2% horse serum, and 2% goat serum, and in primary antibody (unsulfated CS stub antibody, 1B5, mouse IgG1, Seikagaku Corporation) at 4°C overnight. The 1B5 antibody recognizes a disaccharide neoepitope generated at the non-reducing terminal of CS chains that were pre-digested with Chase ABC [20 (link)]. After three washes with Tris-buffered saline, the cells were then treated with Alexa Fluor 488-conjugated anti-mouse IgG1 (Molecular Probes). The nuclei of cells were counterstained with 4’,6’-diamidino-2-phenylindole (DAPI, Sigma).
For CD133 antibody, after incubation in blocking solution containing 10% donkey serum at room temperature for 1h, cells were incubated in primary antibody (CD133, rabbit, Abcam) at 4°C overnight. After three washes, the cells were then treated with Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch) and DAPI (Sigma).
Quantification of immunocytochemical staining was described inS1 File .
For CD133 antibody, after incubation in blocking solution containing 10% donkey serum at room temperature for 1h, cells were incubated in primary antibody (CD133, rabbit, Abcam) at 4°C overnight. After three washes, the cells were then treated with Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch) and DAPI (Sigma).
Quantification of immunocytochemical staining was described in