Qwin standard

Tissue sections (5 µm) were quenched with 3% H₂O₂ for 10 min at room temperature after dewaxing and antigen retrieval in hot citrate buffer. After blocking with 5% BSA for 30 min at 37 °C, tissue sections were incubated with a monoclonal anti-Ki-67 nuclear antigen antibody (ZSGB-BIO) at 4 °C overnight. Immunostaining was assessed in 3 randomly selected fields under a microscope with a 200 × objective lens and photographed. Images were analyzed using a microscope-matched analytical software (Leica QWin Standard).

Sections were de-paraffinized and pre-treated by boiling in 10 mM citrate buffer, pH 6.0, or in high pH target retrieval solution (DAKO). After quenching in 3% H₂O₂, slides were blocked in 20% serum species-matched to the secondary antibody. Staining was developed using DAB (Vector Laboratories, Burlingame, CA) or NBT/BCIP (Roche, Basel, Switzerland). Collagen fibers were visualized using 0.1% Sirius Red. Stereological quantification of capillary tumor blood vessels was performed after CD31 and ASMA, PDGFRβ, NG2 or desmin staining, using an eyepiece grid for unbiased counting, as described earlier [32 (link)]. Stereological quantification of CD31-positive vessels was performed using Leica QWin Standard digital image software and values for 10–40 fields of vision (0.09 mm²) were averaged. The fraction of cleaved caspase-3 positive cells or Ki67 positive cells was determined after analyzing 1000 cells from all tumors in each group.

Histomorphometry of jejunum was carried out at day 24. For this purpose, the
abdomens were opened, intestines were removed, and 3 cm of jejunal samples was
dissected from the midpoint of jejunum towards proximal direction (towards
duodenum) after the identification of Meckel's diverticulum as a reference
point. The jejunal tissues were immediately washed, immersed in 10 %
neutral buffered formalin for fixation, soaked in ascending alcohol
concentrations (70 %, 80 %, 96 %, and 100 %) for dehydration,
cleared in xylene, and finally embedded in paraffin blocks. Sections were
obtained, stained with hematoxylin and eosin and periodic acid–Schiff, and
examined under light microscope (BX51, Olympus, Japan), and images were
produced using a digital camera (SC180, Olympus, Japan). Villus height,
villus diameter, and villus width were measured using a computer-assisted
image analysis program (Leica QWin Standard, Version 2.8, Germany) following
the procedures previously described by Ahsan et al. (2018). Villus height
to crypt depth ratio was measured by division method, whereas villus surface
area was computed according to de los Santos et al. (2005) using following
Eq. (1):
$$\begin{array}{rl}\text{Villus surface area}& =\mathrm{2}\mathit{\pi }\times \left(\text{villus width}/\mathrm{2}\right)\\ & \times \left(\text{villus height}/{\mathrm{10}}^{\mathrm{6}}\right).\end{array}$$

After dewaxing and antigen retrieval, tissue sections were quenched with endogenous peroxidase blockers for 10 min at room temperature. After being blocked with 5% bovine serum albumin (in PBS) for 30 min at 37 °C, sections were incubated with anti-Ki67 (ZSGB-BIO, Beijing, China) at 4 °C overnight. A universal DAB detection kit (ZSGB-BIO, Beijing, China) was used to stain Ki67 antibody bound tissues. Immunostaining was observed in 3 randomly selected fields under a microscope (Leica QWin Standard). The images were photographed and analyzed with Leica QWin analytical software. The percentage of cells staining positive for Ki67 expression were normalized to the values in the control group.