Pannoramic 250 flash scanner

Tissue samples were fixed with 10% formalin, embedded in paraffin, and sectioned to an 8 μm thickness slide. Hematoxylin and eosin (HE) stain and Masson’s trichrome stain were then used to assess the extent of tissue fibrosis. In addition, heart slides were stained with Sirius Red and liver slides were stained with Oil Red to appraise hepatic steatosis. Images were captured using a Nikon Eclipse E100 ortho optical microscope (Nikon, Japan). For IHC, paraffin sections were dewaxed and rehydrated. Antigens were retrieved with citric acid antigen repair buffer and then put into 3% hydrogen peroxide solution to eliminate endogenous peroxidase. The slides were then blocked with 3% BSA and incubated with anti-TNF-α or F4/80 antibody (1:100) at 4 °C overnight. After incubating with HRP-labeled secondary antibody (1:1,000, Servicebio, Wuhan, China), the samples were detected using DAB staining. Images were acquired and analyzed using a Pannoramic 250 FLASH scanner (3D HISTECH, Budapest, Hungary).

Immunoblotting was performed as described previously [25 (link)]. Primary antibodies against AKT (Cell Signaling; 9272S, 1 : 1000, Frankfurt, Germany), phospho‐Akt S473 (Cell Signaling; 9271, 1 : 1000), PME‐1 (Santa Cruz Biotechnology; sc‐20086, 1 : 1000, Heidelberg, Germany), phospho‐PDHE1α S300 (Merck Millipore; ABS194, 1 : 1000, Darmstadt, Germany), cleaved PARP1 (Abcam; ab32064, 1 : 1000, Cambridge, UK), SV40 T Ag (Pab 108) (Santa Cruz Biotechnology; sc‐148, 1 : 1000), β‐actin (Sigma‐Aldrich; A1978, 1 : 10 000), and GAPDH (HyTest; 5G4cc, 1 : 10 000, Turku, Finland). Secondary antibodies were purchased from LI‐COR Biotechnology, Bad Homburg, Germany or Dako (Agilent Technologies, Winooski, VT, USA. The histological methods were performed by the Histology core facility of the Institute of Biomedicine, University of Turku, Finland. Brain sections were stained with phospho‐ERK1/2 (Thr202/Tyr204) (Cell Signaling; #9101, 1 : 500) and Ki67 (Dako; M7240, 1 : 500). Slides were scanned using a Pannoramic 250 Flash scanner (3DHISTECH, Budapest, Hungary), and images were acquired using a slideviewer v2.6 (3DHISTECH).

Each section was scanned using the Pannoramic 250 Flash Scanner (3DHistech, Budapest, Hungary). All the scanned images were analyzed by Image-Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The magnification was 20× for HE sections and 20×/100× for IHC staining. The positive staining in images was quantified as integral optical density (IOD)/area, that is, mean density = density sum/area sum [12 (link)]. In detail, five fields with the strongest expression of α2δ1 were chosen from each section and the average value of mean density was calculated.

IHC analysis was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using a Discovery XT processor (Ventana Medical Systems). Three hours after the last drug treatment, tumors were immediately fixed in fresh 4% paraformaldehyde rotating at 4 °C overnight. Fixed tissues were dehydrated and embedded in paraffin before 5 µm sections were put on slides. The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems) and incubated with antibody against stated antibodies for 5 h, followed by 60 min of incubation with biotinylated mouse secondary (Vector Labs, cat. BMK-2202, MOM in 5.75 ug/mL (1:200)). Slides were scanned with Pannoramic 250 Flash scanner (3DHistech, Hungary) using 20X (0.8 NA) objective lens. Tunel assay for mouse organs was performed following standard protocols at Histowiz, Inc (Brooklyn, NY).

Three hours after the last drug treatment, tumors were immediately fixed in fresh 4% paraformaldehyde rotating at 4 °C overnight. Fixed tissues were dehydrated and embedded in paraffin before 5 μm sections were put on slides. The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems) and incubated with antibodies against Ki-67 (DAKO, cat. M7240, 0.5 µg /mL), or c-MYC (Cell signaling, cat. 13987, 4 µg/mL); BCL-2 (Ventana-Roche, cat. 790-4604, 0.2 µg/mL) for 5 h, followed by 60 min of incubation with the corresponding biotinylated secondary antibodies. Slides were scanned with Pannoramic 250 Flash scanner (3DHistech, Hungary) using 20X (0.8 NA) objective lens.

IHC and ISH staining were performed to quantify expression of the MITF protein and miR-585-5p, respectively. GC tissue microarrays were immuno-stained for MITF (Abcam, # ab270262). The digoxin-labelled nucleic acid probe for miR-585-5p was developed through GenePharma using the reverse complement of the following sequence: 25-CUAGCACACAGAUACGCCCAGA-46. The microarrays were observed/imaged through Pannoramic 250FLASH Scanner (3DHISTECH). IHC and ISH staining were concomitantly assessed through two blinded observers for individual clinical case clinico-pathological profiles. H-scoring was adopted based upon intensity and extent of staining by an experienced pathologist, and graded as: 0, negative staining; 1+, weak staining; 2+, moderate staining; and 3+, strong staining. The H-score was computed by multiplying the different intensities in 4 gradations with each percentage of positive tumour cells: H-score = 1× (% cells 1+) + 2× (% cells 2+) + 3× (% cells 3+). Finally, a score from 0 to 300 points was obtained (13 (link)). Median H-score within cohort was applied as a cut-off for distinguishing high- and low-expression.

Quantification of steatosis was performed with a python script based on HE staining and adapted from scikit-image [33 (link)]. Sagittal sections of a whole liver lobe were included in the analysis. Whole slide images were scanned using a PANNORAMIC 250 Flash scanner (3DHISTECH Kft., Budapest, Hungary). The nucleus and vacuoles of steatosis were automatically detected and validated visually. The relative area of steatosis was calculated as the area of vacuoles normalized by the total area of the whole slide.