Panoramic scan instrument

Apoptosis assays were performed using a colorimetric TUNEL apoptosis assay detection kit (C1098, Beyotime, China) according to the manufacturer’s instructions. Briefly, after the tissue section were rehydrated and rinsed with distilled water, the proteinase K solution was used for antigen retrieval. Then the peroxidase blocking reagent was used to block the endogenous peroxidase. The sections were incubated in the TUNEL reaction mixture at 37 °C for 60 min in the dark, then incubated with Streptavidin-HRP for 30 min at room temperature. Chromogenic detection was performed with DAB chromogenic reagent, and finally, the tissue sections were stained with hematoxylin staining solution. 3DHISTECH panoramic SCAN instrument was used for image acquisition; TUNEL-positive cell nuclei were stained brown, and the negative normal cell nuclei were stained blue. Apoptosis index was calculated as the percentage of TUNEL-positive cells to the total number of cells.

The TMA blocks were cut into 3 µm sections and then applied to silane-coated adhesive microscope slides. Immunohistochemical staining was performed on the Dako Agilent Autostainer System (Agilent, Santa Clara, CA, USA). Antigen retrieval was performed using Target Retrieval Solution, Low pH. TMA sections were labeled with VDR antibody (Santa Cruz, mouse monoclonal antibody, 1:600 dilution). External controls (cerebral cortex, cerebellum, lymph node, liver, kidney, uterus and skin) were used as positive and negative controls. TMA slides were scanned using a Panoramic scan instrument (3D HISTECH) equipped with a 20× Carl Zeiss objective (NA = 0.83; Carl Zeiss MicroImaging Inc., Jena, Germany).
The relative proportion of positive cells was determined on the scanned slides at 400× magnification by counting 200 cells in three different areas on each TMA core by two independent pathologists, evaluating a minimum of a 0.1 mm² area. According to the expression level of the VDR protein, the following scores were assigned: negative < 10% (1—low), 11–50% (2—intermediate) and 51–100% (3—high).
All tissue samples were handled in a coded fashion, according to the Dutch and Hungarian National Ethical guidelines (National Ethical Review Board approval: TUKEB no. 7/2006).

Slides were digitalized using a high resolution Panoramic Scan instrument (3DHistech Ltd., Hungary) and analyzed with the Panoramic Viewer Histoquant Module software (3DHistech Ltd., Hungary). 1000 cells/slide were evaluated. In the case of E-cadherin, CK20, cytoplasmic immunoreactions were scored as negative (-), weak (+), moderate (++) and strong (+++). NFκB, DNMT3a nuclear/cytoplasmic expressions were scored as negative (-), weak (+), moderate (++) and strong (+++) immunoreactions. The density of negative (-), weak (+), moderate (++) and strong positive (+++) pixels in immunoreactive cells was evaluated.