The entire colon was removed from the cecum to the anus, fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 4-μm sections. After hematoxylin and eosin (H&E) staining, histological analysis was performed in a blind manner. The following three parameters were studied as described previously:22 (link) inflammation severity (0=none, 1=mild, 2=moderate, and 3=severe), inflammation extent (0=none, 1=mucosa, 2=mucosal and submucosa, and 3=transmural), and crypt damage (0=none, 1=basal 1/3 damaged, 2=basal 2/3 damaged, 3=crypts loss, but surface epithelium present, 4=both crypts and surface epithelium loss). Total histological score was defined as the sum of the three parameters.
Immunohistochemical (IHC) staining for GFP, using a Vecastain ABC kit (Vector Laboratories, Burlingame, CA, USA), was performed to identify the in vivo localization of transplanted BM-MSCs in the inflamed colon. Colonic tissue samples were incubated first with the primary anti-GFP antibody (Abcam, Cambridge, MA, USA) overnight at 4°C, then with a biotinylated secondary linking antibody, and finally with a streptavidin-peroxidase complex for 1 hour. The final color product was developed using aminoethylcarbazole (Dako, Glostrup, Denmark). Sections were counterstained with hematoxylin, and tissues were photographed using an Olympus photomicroscope (Olympus Corp., Tokyo, Japan).
Immunohistochemical (IHC) staining for GFP, using a Vecastain ABC kit (Vector Laboratories, Burlingame, CA, USA), was performed to identify the in vivo localization of transplanted BM-MSCs in the inflamed colon. Colonic tissue samples were incubated first with the primary anti-GFP antibody (Abcam, Cambridge, MA, USA) overnight at 4°C, then with a biotinylated secondary linking antibody, and finally with a streptavidin-peroxidase complex for 1 hour. The final color product was developed using aminoethylcarbazole (Dako, Glostrup, Denmark). Sections were counterstained with hematoxylin, and tissues were photographed using an Olympus photomicroscope (Olympus Corp., Tokyo, Japan).