Aperio imagescope software

RNA was extracted from full thickness mucosa or LSM as previously described^{13 (link)}. Paraffin embedded full thickness mucosa was used in immunohistochemistry as previously described^{13 (link)}. Infiltrating intact eosinophils and eosinophil degranulation (i.e., the presence of free cytoplasmic granules and/or tissue deposition of eosinophil granule proteins) were assessed by immunohistochemistry using a mouse monoclonal anti-eosinophil peroxidase antibody (EPX-mAb) as previously described^{38 (link),39} . Tissue sections were digitized (Aperio AT Turbo, Leica Biosystems, Buffalo Grove, IL) and images were generated using Aperio ImageScope software (version 11.2.0.780, Aperio Technologies, Vista, CA).

After sacrifice, isolated zinc-fixed carotid and femoral arteries were dehydrated and embedded in paraffin. Serial cross-sections (3 μm) were cut and histomorphometric analysis was performed at 20 cross-section levels (with 66 μm intervals) (Fig. S1).
Sections at each level were stained with Verhoeff-van Gieson or Masson’s trichrome for collagen content and slide images captured using a Hamamatsu Nanozoomer 2.0HT (Hamamatsu Photonics, Hamamatsu, Japan). Morphometric analysis was performed using Aperio Imagescope software (Aperio Technologies inc, CA, USA). Percentage intimal expansion throughout the arterial segment was calculated with the following formula: % intimal expansion = ((A_I − A_L)/A_I)) × 100; where A_I is the area within the internal elastic lamina and A_L is the luminal area. The intima/media (I/M) ratio was calculated with the following formula: I/M ratio = (A_I − A_L)/(A_M − A_I); where A_M is the area within the external elastic lamina. The media and adventitial areas were also quantified. To determine loss of cells after vascular injury, the total number of cells in media and adventitia were quantified. Serial sections, as depicted in Fig. S1, were quantified to determine the mean % intimal expansion per artery. The number of sections analyzed was an average of 12 (range 9–17) per injured artery and an average of 4 sections (range 3–5) per control artery.

Paraffin-embedded tissue sections and TMAs underwent IHC analyses. Briefly, the slides were probed with primary antibodies specific for the following proteins CBX6, PCNA, and Ki67, and then the slides were treated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Finally, the slides were stained with diaminobenzidine (DAB) colorimetric reagent solution from Dako (Carpinteria, CA, USA) before undergoing hematoxylin counterstaining (Sigma Chemical Co). TMA analysis was performed by scanning the slides with an AperioScanScope GL, and AperioImageScope software (Aperio Technologies, Vista, CA, USA) was used to assess the scanned images by determining the percentages of positively stained cells and staining intensities. CBX6expression in all the clinical samples was quantified, and the tumor CBX1-8 expression level/peri-tumor CBX1-8 expression level ratio was calculated.

Sections (4 μm) of paraffin-embedded samples were incubated with anti-AEG-1 (1:300) (Abcam, Cambridge, UK), anti-E-cadherin (1:200) (Abcam, Cambridge, UK) or anti-vimentin (1:250) (Abcam, Cambridge, UK) primary antibodies. We applied the known positive slice in the SP kit (Maxim-Bio, Fuzhou, China) as a positive control. Sections developed using 3, 3”-diaminobenzidine as the chromogen and hematoxylin as the counterstain. The numbers of positive cells were semi-quantitatively evaluated under a light microscope. The staining index was calculated using Aperio ImageScope software (Aperio Technologies).