Aperio imagescope v12

A total of 205 WSIs were acquired from 205 pathology slides with an Aperio AT2 slide scanner (Leica Biosystems Division of Leica Microsystems Inc., IL, USA) and 400 ×. Two experienced pathologists annotated the cancer regions on the WSIs with Aperio ImageScope v12.4.3 (Leica Biosystems Division of Leica Microsystems Inc., IL, USA). Tumor areas were extracted from the annotated whole slide images (WSIs), the extracted areas were used to generate non-overlapping patches 256 × 256 pixels in size at a magnification of 20 × using DeepPATH based on the OpenSlide library in Python (Fig. 1a)^{7 (link)}. The patches were removed if the percentage of background in the patch was above 25% according to the DeepPATH^{7 (link)} program. In this process, 10,049 patches were generated from the original 205 WSIs containing either one of the four lung cancer subtypes or a negative case, as shown in Table 1.

Slides were imaged using a Nikon Eclipse E800 microscope with NIS-Elements Basic Research Software or by the Vanderbilt University Medical Center Digital Histology Shared Resource (DHSR). For chromogenic IHC, whole slides were imaged by the DHSR using a Leica SCN400 Slide Scanner (Leica Biosystems). For fluorescence staining, whole slides were imaged by the DHSR using an Aperio Versa 200 automated slide scanner (Leica Biosystems) and visualized using Aperio ImageScope (v12.4.3) (Leica Biosystems). Quantification was performed, according to a protocol blinded to genotype, by counting positive cells in well-oriented crypts or crypt-villus units.

The immunohistochemistry staining of EpCAM was performed at MSK Molecular Cytology Core Facility, using a Discovery XT processor (Ventana Medical Systems). Paraffin-embedded PDX sections derived from GC patient were kindly provided by MSK Antitumor Assessment Core Facility. A mouse anti-human EpCAM monoclonal antibody (Agilent, clone Ber-EP4) was used. Stained slides were scanned by HistoWiz and analyzed using Aperio ImageScope v.12.4.3 (Leica Biosystems).

These values were obtained with the annotation tool on the software Aperio ImageScope v.12.4.3 (Leica Biosystems) on histological slides. The free-form drawing tool was applied by coloring the borders of each nerve fiber where this action also automatically created a table with the number of structures selected along with its corresponding diameter in mm and area in mm². Total nerve area (TNA) is defined as the sum of each nerve fiber area value at the level of the middle colic artery origin within the D3 area. SMA sheath thickness in Group II specimens was measured with the digital ruler tool in ImageScope software.

Flu infected and control mice were euthanized by CO₂ asphyxiation, and their tracheas cannulated with a 20 G flexible catheter (Surflo, Terumo, Philippines). The lungs were gently lavaged with 600 µl of PBS in four passes. The supernatant from the first pass was collected and used for further analyses. The cells from all four passes were pooled and re-suspended in 1 ml of PBS and counted using a Nexcelcom cell counter. Lungs were fixed by inflation with 10% buffered formalin at 20 mm H₂O of pressure, paraffin embedded, and stained with H&E stain and PAS. Stained slides were digitally scanned at ×63 magnification using an Aperio CS-O slide scanner (Leica Biosystems, Chicago IL). Representative images were taken from scanned slides using Aperio ImageScope v12.2.2.5015 (Leica Biosystems, Chicago, IL). The histological and cytological scoring were performed in a blinded fashion. Numerical codes were used to identify these slides during the scoring. Once all the data were recorded, the identity was unmasked and final analyses undertaken according to the study group.