Pannoramic p250

During review, histological slides containing the most representative areas of normal colonic tissue, tumour centre and tumour invasive front were selected. Corresponding blocks were retrieved from the archives of the Institute of Pathology (University Hospital Erlangen). Slides were scanned (Pannoramic P250, 3DHistech, Hungary). Each slide was then viewed and annotated using a TMA annotation tool (Panoramic viewer v15.1. and the corresponding TMA annotation tool, 3D Histech, Hungary) 7, 9. We chose a diameter of 0.6 mm, which reflects the area of a high power field (HPF) of a standard microscope (eg, field‐of‐view number 23–24, aperture 0.575–0.625 mm). Each region was annotated in duplicate or triplicate as depicted (Figure 1).
To achieve a TMA for tumour microenvironment studies we placed the marking in such a way that cores from the tumour centre contain a maximum ratio of epithelium to stroma, ie as many tumour epithelial cells as possible from each case, and that cores from the tumour invasive front included 50% epithelium and 50% stroma (Figure 1a–d).
Once all scanned slides were reviewed, the corresponding donor blocks were loaded into an automated tissue microarrayer (TMA Grandmaster, 3DHistech, Hungary) and aligned. In total, 13 ngTMAs were constructed.

The xenograft and allograft were fixed in 10% phosphate-buffered formalin,
embedded in paraffin, and cut into 5 µm sections for hematoxylin and eosin
(H&E) staining. Immunohistochemical staining was performed as described previously^{17 (link)}
, the sections were then stained with primary antibody anti-CD4 Ab
(Boster, Wuhan, China), anti-CD8 Ab (Boster), anti-CD20 Ab (Boster), anti-IgM
(Boster), and secondary antibody goat anti-rabbit IgG (GB23303; Boster, Wuhan,
China). Samples were visualized with a DAB detection kit (Maixin-Bio, Fuzhou,
China). We used a pathological section scanner (Pannoramic P250, 3DHISTECH,
Budapest, Hungary) to analyze the immunohistochemical staining density. Two
cardiologists blinded to the experimental conditions graded acute rejection
according to the International Society of Heart and Lung Transplantation (ISHLT) criteria^{21 (link)}
. Briefly, 0 R = no rejection; 1 R (mild rejection) = evidence of
perivascular infiltrate, interstitial infiltrate, or both with up to 1 focus of
myocyte damage; 2 R (moderate rejection) = two or more infiltrate foci with
related myocyte damage; 3 R (severe rejection) = the infiltrate was diffuse and
had multifocal myocyte damage ± edema, ± hemorrhage, ± vasculitis.

A TUNEL assay was performed to measure cell apoptosis using the In Situ Cell Death Detection Kit, Fluorescein (Roche Molecular Diagnostics, Pleasanton, CA, USA). Briefly, transfected PC9R cells (5×10⁵ cells/well) were seeded on glass coverslips coated with poly-L-lysine (ScienCell Research Laboratories, Inc., San Diego, CA, USA) and treated with 1 µM gefitinib. Following transfection for 96 h, cells were fixed in 3.7% formaldehyde in PBS for 15 min at room temperature. TUNEL staining was performed according to manufacturer's protocol. After TUNEL staining, DAPI staining (Thermo Fisher Scientific, Inc.) was performed. Slides were scanned (Pannoramic P250; 3DHistech Ltd., Budapest, Hungary) and viewed using the Pannoramic Viewer software (3DHistech Ltd.). PC9R cells positive for TUNEL and DAPI staining were counted using ImageJ software (version 1.42), and the percentage of TUNEL-positive cells was calculated.