Nanozoomer digital pathology system nanozoomer 2.0 ht slide scanner

For immunoenzymatic staining, spleens were collected. Formalin fixation, paraffin inclusion, as well as standard staining (hematein-eosin) were performed on this tissue. Immunohistochemical staining was performed on 3‐µm–thick tissue sections from the formalin‐fixed paraffin‐embedded spleen specimens, by a standardized automated method (Bond; Leica Menarini) using anti-CD20 M0755 (L26 clone, Dako, anti-CD5, NCL-L-CD5-4C7, 4C7 clone, Leica Biosystems), anti-CD38 (NCL-L-CD38-290, SPC32 clone, Leica Biosystems), anti-CD138 (M7228, MI15 clone, Dako, anti-CXCL13, MABB801, clone 53610, R&D systems), anti-BCL6 (PA0204, LN22 clone, Leica Biosystems), anti-ICOS (ab105227, SP98 clone, Abcam), and anti-PD1 (ab52587, NAT105 clone, Abcam). Slides were scanned using NanoZoomer Digital Pathology System (Nanozoomer 2.0-HT slide scanner (Hamamatsu, Hamamatsu City, Japan)). Cell detection was conducted using QuPath’s digital software (https://qupath.github.io) built-in ‘Positive cell detection’. Cell densities of immune cells were expressed as the mean number of positive cells per mm².

The RCCEP lesion tissues were fixed with 10% neutral formaldehyde, dehydrated with an alcohol gradient, infiltrated with paraffin wax, and embedded in paraffin to form paraffin-embedded blocks. Sections (4 μm) were cut for hematoxylin and eosin (H&E) staining, immunohistochemical (IHC), and immunofluorescence staining (Multiplex IF). The sections were baked at 60 °C overnight, and then IHC and Multiplex IF staining were carried out using a Bond RX automatic stainer (Leica Biosystems, Buffalo Grove, IL). The stained sections were scanned using NanoZoomer Digital Pathology System (Nanozoomer 2.0-HT slide scanner (Hamamatsu, Hamamatsu City, Japan). The NDP view imaging software (NDP scan software) was used to analyze the scan results and catch representative images.

Tissue specimens were harvested immediately after IRE and 24 h, 3 d, 7 d, 14 d, 28 d and 6 mo (N = 3 for each time point up to 6 months and N = 6 for a 6 months time point) following the initial IRE ablation. Liver samples were fixed in 10% formalin, embedded in paraffin, and cut into 5 μm sections. Sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome stains by the Pathology Core at the Massachusetts General Hospital. Slides were evaluated by three separate investigators. Color images of each entire tissue section were acquired using NanoZoomer Digital Pathology System (Nanozoomer 2.0-HT slide scanner; Hamamatsu, Hamamatsu City, Japan).