Camedia software

Colorectal sections were stained with H&E. For immunohistochemistry, paraffin-embedded colorectal tissue sections were autoclaved for 2 minutes in citrate buffer to promote antigen retrieval. After blocking in 5% bovine serum albumin, sections were incubated with primary antibodies against Lgr5 (1:100, Abcam) and Cyclin D1 (1:100, Abcam) at 4°C for overnight. Sections were then incubated with secondary antibodies for 30 minutes at 37°C, visualized with Diaminobenzidine (DAB) (Dako Denmark A/S, Glostrup, Denmark), and then counterstained with hematoxylin and observed under a light microscope using Olympus Camedia Software. Two pathologists analyzed the expressions of Lgr5 and Cyclin D1 in a blinded manner. For quantification of the immunohistochemical staining, relative protein expression of Lgr5 and Cyclin D1 was quantified by Image Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) and was defined as follows: relative protein expression=IOD/ area sum, where IOD represents integrated OD.

Tumor sections were stained with TUNEL kit or antibodies according to the manufactory protocol. Sections were visualized with DAB (Dako, Denmark), and then counter-stained with hematoxylin and observed under a light microscope using Olympus Camedia Software.

Paraffin-embedded sections (4 µm thick) on polylysine-coated slides were used for staining. Antigen retrieval was achieved with 10 mmol/L sodium citrate (pH 6.0). Thereafter, sections were blocked with 10% normal goat serum and then incubated with rabbit monoclonal antibody against FTO (EPR6895, Abcam, 1:1000) overnight at 4 °C. After incubation with secondary antibody (Vector Laboratories), staining was performed using the DAB Substrate Kit for Peroxidase (Vector Laboratories). FTO expression was evaluated by the immunodetection of staining intensity and the number of positive cells. Photomicrographs were imaged using an Olympus BX53 microscope with plan apochromatic objective lenses. An Olympus DP11 camera and Olympus Camedia software were used to produce the images. All cases were reviewed by two pathologists who were blinded to the study and the images were quantified using Image Pro Process software.