Olyvia v3

Peritoneal biopsies were formalin-fixed in 4% buffered formalin and paraffin-embedded following routine protocols [29 (link)]. Immunohistochemistry was performed as previously described [28 (link),30 (link),31 (link)]. Used antibodies: anti-TXNIP (ab188865; 1:200, Abcam, Cambridge, UK), anti-TRX (sc-271281; 1:100, Santa Cruz), anti- γH2AX (9718S; 1:100, CST).
For morphology, slides underwent hematoxylin and eosin (HE) staining. Tissue sections were examined with Olympus VS120 automated slide scanner equipped with a BX61VS microscope (objective: UPLSAPO 20× or UPLSAPO 2 40×, Olympus, Tokyo, Japan). Mesothelial cell loss was evaluated as: completely contained (0); partially lost (1); completely lost (2). Mean submesothelial thickness was evaluated by measuring the thinnest and thickest layer using the imaging software (OlyVIA V3.3, Olympus).

Cytospun cells were fixed using 4% neutral buffered formalin (Sigma-Aldrich, Missouri, MO) following by permeabilization with 0.4% Triton X-100. For background blocking, cells were incubated for 15 min with Background Sniper (Biocare Medical, Pacheco, CA) followed by 1 h with 5% bovine serum albumin (BSA; Sigma-Aldrich). The samples were incubated with anti-vimentin human-specific primary antibody (V9; cat no. 790-2917; Ventana, AZ) and an antibody cocktail against cytokeratins 8, 18 and 19 (anti-KRT8 (HPA049866), anti-KRT18 (HPA001605), anti-KRT19 (HPA002465); Sigma-Aldrich) overnight, followed by secondary antibodies labelled with Alexa Fluor™ 647 and Alexa Fluor™ 488 (Invitrogen, Waltham, MA), respectively, for 2 h. The slides were covered in mounting solution mowiol and coverslips added. Slides were scanned using VS200 Olympus Slidescanner at ×20 magnification (40 × 0.27 µm resolution), and images collected and assessed using OlyVIA V3.3 (Olympus, Japan). Total CTCs were determined from the combined sum of all stained cells.

After the dielectric measurement procedure was completed, seven samples of tissue used in MWA experiments in TMD Lab (1 cm to 2 cm wide and less than 5 mm thick) were taken from three lung samples (2 inflated, 1 not inflated) and embedded in formalin; three samples were taken from inside the ablation zone; three from not ablated tissue, and one included both ablated and not ablated tissue. For each sample it was noted the parent lung sample, if it was subject to inflation and ablation. Samples were embedded in formalin 2 to 4 h after ablation end.
The tissues were transferred into 4% paraformaldehyde (PFA) in PBS (pH.6.9) and stored at room temperature for at least 24 h until processing and embedding³¹ . The tissues were then processed in series of alcohols and embedded in Paraffin using Excelsior ES tissue processor (A82310100, Thermo Scientific, Waltham, MA, USA) and HistoCore Arcadia H embedding system (14039357258, Leica Biosystems, Wetziar, Germany).
The paraffin-embedded blocks were sectioned to obtain 5 µm sections and stained with haematoxylin and eosin (H&E), dehydrated, and mounted with dibutylphthalate polystyrene xylene (DPX). Images of the slides from H&E were acquired using Olympus Virtual Slide Scanner VS120-L100-W and OlyVia v3.3 (Olympus Life Science Waltham, MA, USA).