Nanozoomer s60

Parts of VAT, SCAT, and liver were fixed in 4% paraformaldehyde (Sigma-Aldrich) overnight at 4 °C, dehydrated and embedded in paraffin as previously described [32 (link)]. Tissue Sections (5 μm) were prepared by using a microtome and stained with hematoxylin and eosin (Sigma-Aldrich). VAT and SCAT were acquired using the Axiovision Zeiss software (10 × magnification). The quantification of adipocytes area was performed using ImageJ software with the Adiposoft plugin [33 (link)]. For the liver section, whole slices were acquired with the Nanozoomer S60 slide scanner (Hamamatsu, Tokyo, Japan). The images were then magnified and saved at 20 × with the NDPview2 software (Hamamatsu) to show only the considered area, which was then quantified with ImageJ software. Area covered by small lipid droplets within hepatocytes was evaluated as microsteatosis, whereas area covered by large lipid droplets disrupting the cell morphology (small nuclei pushed to the cell border) was quantified as macrosteatosis [34 (link)]. Inflammatory foci were evaluated as groups of > 5 close non-hepatocytic nuclei [35 (link)].

Immunohistochemical staining with monoclonal mouse anti-human mitochondria (hMIT) (clone 113-1, Merck Millipore, Burlington, MA, 1:2000), antihuman CYP1A2 (clone 3B8C1, Abcam plc., Cambridge, UK, 1:1000), antihuman CYP2C9 (clone 2C8, LifeSpan Biosciences, Inc., Seattle WA. USA, 1:150), and rabbit antihuman CYP3A4 (clone EPR6202, Abcam Plc., 1:300) antibodies was performed as described previously^{14 (link)}. Tissues were fixed in 4% (v/v) phosphate-buffered formalin (Mildform 10 NM; Wako Pure Chemical Industries). The sections were autoclaved for 10 min in a target retrieval solution (0.1 M citrate buffer, pH 6.0; 1 mM EDTA, pH 9.0), equilibrated at room temperature for 20 min, and then incubated with an anti-POR (HPA010136, Sigma) primary antibody. Primary antibodies were visualized using amino acid polymer/peroxidase complex-labeled antibodies (Histofine Simple Stain MAX PO [MULTI]; Nichirei Biosciences Inc.) and diaminobenzidine (DAB; Dojindo Laboratories, Kumamoto, Japan) substrate (0.2 mg/mL 3,3-diaminobenzidine tetrahydrochloride in 0.05 M Tris–HCl, pH 7.6, and 0.005% H₂O₂). The sections were counterstained with hematoxylin. Images were captured using a digital slide scanner (NanoZoomer S60; Hamamatsu Photonics, KK Hamamatsu, Japan).

TUNEL was used to detect the apoptosis of intestinal epithelial cells. TUNEL staining was processed by an apoptosis two-step assay kit. The slides of the TUNEL stain were sealed by an anti-fluorescence quenching sealer. The images of the slides above were collected by NanoZoomer-S60 (Hamamatsu Photonics (China) Co., Ltd.) and analyzed by ImageJ.

With each rat under general anesthesia, 10 $\mathrm{\mu }$ L of ICG aqueous solution (50 µg/mL) or SnC–ICG (final ICG concentration: 50 µg/mL) was administered to the right foot pad of four animals each and the injection site was massaged a few times. Eighteen hours later, each rat was laparotomized and regional LNs were observed using an NIR fluorescence camera. Next, all fluorescent LNs were retrieved and embedded in a Tissue-Tek® Optimal Cutting Temperature Compound (O.C.T. compound; Sakura Finetek Japan Co., Ltd., Inc., Tokyo, Japan) and subsequently frozen in liquid nitrogen.
Tissue sections (7  $\mathrm{\mu }$ m thick) were prepared and the distribution of fluorescence in the specimen was examined under the NIR fluorescence microscope. Next, the samples were stained with hematoxylin/eosin (HE) and their images were obtained using a digital slide scanner (NanoZoomer S60; C13210-01, Hamamatsu Photonics K.K., Shizuoka, Japan).
The fluorescence intensity of the LN images on the NIR fluorescence camera was estimated using the ImageJ software (NIH, DC, USA). The mean brightness of the fluorescent area determined by a five-time repeated discriminant analysis in the CIE Lab color space on an apparent LN image was used as the representative fluorescence intensity.

Staining of formalin-fixed, paraffin-embedded tumors was performed as previously described (16 (link)). Briefly, deparaffinized sections were subjected to heat-induced epitope retrieval (citrate buffer pH6.0), blocked with 5% goat serum in PBS and incubated with a self-made anti-MnPV E4 mouse monoclonal antibody overnight at 4°C. After washing, slides were incubated with Alexa594-conjugated anti-mouse antibody (Invitrogen, Carlsbad, CA, USA) and nuclei were stained with DAPI. Sections were mounted with Dako Faramount Aqueous Mounting Medium (Dako North America, Inc, CA, USA) and imaged with a Keyence BZ-9000 Microscope (Keyence Deutschland GmbH, Neu-Isenburg, Germany) or a Hamamatsu NanoZoomer S60 (Hamamatsu, Hamamatsu City, Japan).

Micronuclei (MNi) multiple MNi (≥2), nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), and fused nuclei (Fused) were scored in binucleated cells using the cytokinesis-block micronucleus cytometry assay (CBMNcyt) [35 (link)]. Briefly, following treatment with MGO for 24 or 48 h, cells were washed twice with Hanks balanced salt solution and resuspended in RPMI-1640 containing 4.5 µg/mL cytochalasin-b (cyt-b) for 24 h. After cytokinesis block, cells were harvested onto slides by cytocentrifugation [35 (link)]. Slide preparation and scoring of the CBMNcyt assay were performed as described previously using a NanoZoomer S60 (Hamamatsu Photonics, Shizuoka, Japan) [35 (link),36 (link)]. Enlarged, misshapen, and multinucleated cells were prepared the same as for the CBMNcyt assay, except cytochalasin-b was not added following treatment of MGO. Scoring criteria for the above biomarkers has previously been described [18 (link),35 (link)]. For mitotic index, cells which possessed condensed chromosomes were distinguished from interphase cells microscopically, as previously described [37 (link)].