Leica aperio cs2

Hematoxylin and eosin staining was performed on tissue sections after MSI analysis. The matrix was removed by immersion in 100% methanol for 30 s, after which the following protocol was used. The tissues were first washed with series of solutions (2 × 95% EtOH, 2 × 70% EtOH and deionized water for 2 min each), stained with hematoxylin for 3 min and subsequently washed with running tap water for 3 min. Tissues were then stained with eosin for 30 s and washed with running tap water for 3 min. After staining, the samples were placed into 100% ethanol for 1 min, then in xylene for 30 s. Finally, glass coverslips were placed onto the samples using Entellen mounting medium. High-resolution optical scans of the stained tissue sections were acquired using a Leica Aperio CS2 (Leica Biosystems Imaging, Vista, CA).

Lung samples were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm sections. The sections were stained with hematoxylin and eosin (H&E) to assess the degree of inflammation. H&E staining was visualized using Leica Aperio CS2 (Leica Biosystems, USA).

TILs were evaluated in 809 whole section formalin-fixed paraffin-embedded (FFPE) tumor tissue samples stained with H&E. The slides were scanned at 200× magnification using a Leica scanner (Leica Aperio CS2, Leica Biosystems, Mount Waverley, VIC, Australia), and the images were visualized using the Aperio ImageScope free software (version 12.3.5.5080).
TILs assessment was independently performed by the senior consultant histopathologist (HO) and one trained researcher (JP). Both were blinded to the clinical data. A consensus decision was made in cases with diverse result.
Briefly, all mononuclear cells within the stroma in the tumor area were considered as sTILs (referred to as TILs) and were estimated as a percentage (1–100%) of the total stromal area within the tumor and its borders. TILs in areas of necrosis, outside of tumor borders, or within in situ carcinomas were excluded. Based on the percentage of TILs, tumor samples were considered to have low (<10%), intermediate (10–39%), or high (≥40%) infiltration. Forty-six samples consisting of in situ carcinoma, microinvasive carcinoma, benign tissue, Paget’s disease, intracystic tumors, or of poor quality preparation were excluded following the exclusion criteria described in the guidelines by the Immuno-Oncology International TILs Working Group (https://www.tilsinbreastcancer.org/) (accessed on 2 February 2021) [37 (link)].