Image pro plus 7

After fixation in 10% formalin, the intestinal tissues were embedded in paraffin wax and cut into 3 μm slices. The slices were then dehydrated with gradient alcohol, cleaned with xylene, and sealed with resin. Hematoxylin and eosin (H&E) staining was performed on the slices. Finally, images were captured using a light microscope (Nikon, Tokyo, Japan) to detect histopathological changes. The severity of intestinal injury was assessed according to Chiu’s scoring system, as previously described [9 (link)].
For immunohistochemistry (IHC), tissue slices were treated with anti-Fas antibody (1:500 dilution; Cell Signaling Technology). The horseradish peroxidase (HRP)-conjugated secondary antibody (Gene Tech, Shanghai, China) was incubated for 30 min at room temperature and used to detect the primary antibody. The images were acquired using a light microscope (Nikon), and Fas staining was quantified using Image-Pro Plus 7 (Media Cybernetics, MD, USA).
Apoptotic cells in the intestinal epithelium sections were detected using TdT-mediated dUTP-biotin nick end-labeling (TUNEL) reagent (Elabscience, Wuhan, China) according to the manufacturer’s instructions. Images were acquired using a fluorescence microscope (Leica Microsystems), and TUNEL-positive cells were quantified using Image-Pro Plus 7 (Media Cybernetics).

For immunohistochemical staining, the sections were deparaffinized in xylene and rehydrated through graded ethanol. Antigen retrieval was performed for 20 min at 95°C with sodium citrate buffer (pH 6.0). After quenching endogenous peroxidase activity with 3% H2O2 and blocking non-specific binding with 1% bovine serum albumin buffer, sections were incubated overnight at 4°C with indicated primary antibodies. Following several washes, the sections were treated with HRP conjugated secondary antibody for 40 min at room temperature, and stained with 3, 3-diaminobenzidine tetrahydrochloride (DAB). Slides were photographed with microscope (Olympus BX43F, Japan). The photographs were analyzed based on the ratio of the staining with the Image-Pro Plus 7.0 software (Media Cybernetics, Inc., Silver Spring, MD, USA). The anti-NCAPG2 used in this manuscript is (ab110882, 1:100).

On the 14th day, five pictures were taken randomly for every culture condition, mSKP spheres were counted, and their diameters were measured using Image-pro Plus 7.0 (Media Cybernetics, USA). Then the number and diameter of mSKP spheres obtained from every culture condition were compared.