Panoramic 250

The expression of α-SMA was analyzed by IHC staining. Sectioned slides were blocked with 5% BSA /PBS at 25 °C for 20 min and then incubated with the anti-ɑ-SMA (1:500) at 4 °C overnight. After incubation with secondary antibodies, the slides were visualized using 3,3′-diaminobenzidine (Dako, CA, USA). The sections were observed using a panoramic microscope (3D HISTECH Panoramic 250, Hungary), and analyzed by ImageJ 1.53a software [39 (link)].

After dehydration and mounting in Permount (Fisher Scientific, Fairlawn, NJ), slides were scanned using Panoramic 250 digital slide scanner (3DHistech, Ltd, Budapest, Hungary) with 20x lens (NA 0.8). Telencephalic areas examined were identified on the basis of the anteroposterior and mediolateral coordinates of Paxinos and Franklin⁶⁷ . Regional structures were confirmed by the distinctive cytoarchitectonic features of the areas in haematoxylin and eosin stained sections. We used Metamorph Software (Molecular Devices, PA) to quantify the immunoreactive elements in 8–12 randomly selected sections from the antero-posterior regions. Each slide included 2–3 sections of each group (WT and PS19, treated and control groups). The first slide was separated by 300 microns from the following slide. Regions of study were designated by using Panoramic Viewer Software (3DHistech Ltd, Budapest, Hungary). Regions were exported into tiled tiff images and used for image analysis. The threshold for positive signal was established for each marker and used identically in all the groups. No difference in total area was identified due to shrinkage of tissue and the intensity was plotted as the average from the total area. The signal was expressed in percent of positive versus total area for each region and case (expressed as mean and standard error of the mean).

For micro-cell-block (MCB) preparation, patient-derived tumoroids corresponding to 3–5 × 10⁴ cells were collected on the day of isolation (D0) and from the 96-well ULA plate at the end of drug screening (D12). Cells were captured in plasma-thrombin clots and fixed, counterstained with Hematoxylin, and embedded in paraffin for sectioning and staining. Embedded material was cut into 2.5-µm-thick serial sections followed by deparaffinization, rehydration and antigen retrieval with the help of an automated immunostainer (Bond RX, Leica Biosystems, Germany). Antigen retrieval was performed in Tris for 30 min at 100 °C for synaptophysin (1:100, 27G12, Novocastra, Leica Biosystem—Deer Park, USA). Primary antibody incubation was 30 min at the specified dilutions. For visualization, a Bond Polymer Refine Detection kit, using DAB (3,3′-Diaminobenzidine), was used as the chromogen. Slides were counterstained with hematoxylin. Scans were acquired with an automated slide scanner Panoramic 250 (3DHistech, Hungary) at 20× magnification. Images were acquired using QuPath software.

For immunohistochemistry formalin-fixed paraffin-embedded patient material was cut in 2.5 µm thick serial sections followed by deparaffinization, rehydration, and antigen retrieval using an automated immunostainer (Bond RX, Leica Biosystems, GER). Antigen retrieval was performed for epidermal growth factor receptor (EGFR) with protease for 5 min at 37 °C. EGFR antibody was diluted 1:25 (Supplementary Table 3). Slides were counterstained with hematoxylin. Scans were acquired with an automated slide scanner Panoramic 250 (3DHistech version 3.0.2) at ×40 magnification. Images were analyzed using the QuPath software^{45 (link)}.

Freshly isolated hearts were immersed in 4% PFA on ice overnight. Tissues were then dehydrated and embedded in paraffin wax. We cut 7-μm sagittal sections through the tissues, making note of serial section number. Sections obtained at similar planes were then used for haematoxylin and eosin staining using a Leica Autostainer (87) (link). Briefly, tissue sections were washed in distilled water followed by staining with Gill’s 3 Hematoxylin to stain nuclei and Eosin Y to counterstain cytoplasmic regions. We imaged stained sections using a Panoramic 250 (3D Histech) slide scanner with CaseViewer software.

Tissue sections were scanned either using a 3DHISTECH Panoramic 250 digital slide scanner (3DHISTECH, Hungary) or with Hamamatsu NanoZoomer S210 Slide-Scanner and the scans analyzed using Definiens® software (Definiens AG, Germany). Bioinformatic analyses were performed in the statistical programming language R (version 3.5.1). All other statistical analyses were performed using Graphpad Prism, San Diego, CA. Statistical tests used, n numbers and P values are displayed in the appropriate figures and figure legends. P values < 0.05 were considered statistically significant.

The fixed kidneys were dehydrated and paraffin-embedded. The samples were then sectioned at 4 µm thickness, dewaxed, and rehydrated. Slides were stained with H&E, PAS, and MT to analyze inflammation, fibrosis, atrophy, and dilation [38 (link)]. The slides were analyzed under a panoramic microscope (3D HISTECH Panoramic 250, Budapest, Hungary).