Nanozoomer xr

Kidneys were fixed in 10% buffered formalin overnight, were processed, and were embedded in paraffin. Sections were stained with Hematoxylin and Eosin (HE), Periodic Acid Schiff (PAS), Periodic acid methenamine silver (PAM) and Mason’s trichrome. All slides were digitally scanned by Hamamatsu Nanozoomer-XR digital slide scanner (Hamamatsu Photonics K. K., Japan) for semiquantitative analysis of histopathological examination. Glomerular changes, inflammatory cell infiltration, and interstitial fibrosis were evaluated and scored based on a 0–3 intervals of 0.5. 0 = no pathology, 1 = mild pathology, 2 = moderate pathology and 3 = severe pathology.

Both the 113 primary CRC tumours with 105 liver and 59 lung metastases were included in analogous immunohistochemical analyses. Formalin-fixed paraffin-embedded whole tissue sections of 3 μm thickness were used. Mismatch repair status was determined by immunohistochemical analysis for the expression of MLH1, PMS2, MSH2, and MSH6 as described previously [47 (link)]. Staining for PD-1 (The Human Genome Organization (HUGO) name PDCD1) and PD-L1 (HUGO name CD274) was conducted with anti-PDCD1 (SP269, 1:50; Spring Bioscience, Pleasanton, CA, USA) and anti-CD274 (E1L3N, 1:100; Cell Signaling Technology, Danvers, MA, USA) antibodies, using a BOND-III stainer (Leica Biosystems, Buffalo Grove, IL, USA). Staining for CD3 and CD8 was conducted with anti-CD3 (LN 10, 1:200; Leica Biosystems, Newcastle, UK) and anti-CD8 (SP16, 1:400; Thermo Scientific, Fremont, CA, USA) antibodies, using a Lab Vision Autostainer 480 (ImmunoVision Technologies Inc., Brisbane, CA, USA). Signal visualization was done by diaminobenzidine and sections were counterstained with haematoxylin. Slides were scanned with a NanoZoomer-XR (Hamamatsu Photonics, Hertfordshire, UK) at ×20 magnification.

Slides were scanned with Hamamatsu Nanozoomer XR using 40× magnification. QuPath software (version 0.2.3) [23 (link)] was used to detect cytoplasmic IGF2BP3 positivity in TMA-sections from annotated areas with leukemic cells. A pathologist manually set detection parameters and thresholds using the cytoplasmic staining of IGF2BP3 in germinal center cells as a reference, and the nuclear staining in germinal centers and proliferating epithelium as a reference for Ki-67 staining. The stain vectors and intensity thresholds for the cell and antibody detection were adjusted according to the instructions of the QuPath software in visual control. Inadequate samples were removed from the analysis. Areas with artifacts caused by compression or folding of the tissue were disregarded by setting the proper threshold values for background intensity. With the IGF2BP3 and CD19/Ki-67- double-stained slides, stain vectors were adjusted for hematoxylin, 3,3′-diaminobenzidine (DAB), and alkaline phosphatase (AP) staining using a representative region of interest. Hematoxylin-stained cells were detected using the cell detection function in the QuPath, while the nuclear DAB of Ki-67-positive cells were recognized from the CD19-positive (AP) areas. Single intensity thresholds for IGF2BP3, CD19, and Ki-67 were used to assess the proportion of positive cells.

The tumors were fixed in formalin and were paraffin-embedded to be sectioned and further processed (Breast Cancer Now Histopathology Core Facility). Multiple sections were taken at regular intervals across each tumor, stained with hematoxylin and eosin (Leica) and an anti-EGFR mAb (Dako pharmDx) according to the manufacturer’s protocol, and imaged with the NanoZoomer-XR (Hamamatsu Photonics).