Nanozoomer slide scanner

Photomicrographs were taken using a Leica DM IL LED Tissue culture microscope (Wetzlar, Germany). Confocal microscopy was performed with a Nikon A1 Upright Confocal microscope (Tokyo, Japan) using 10× or 20× objectives. Imaging of hematoxylin and eosin-stained sections was performed using a Hamamatsu NanoZoomer slide scanner (Hamamatsu City, Japan). Image processing was performed with Nikon Elements, Image J (Fiji) (National Institutes of Health, Bethesda, MD, Photoshop CS6 (Adobe, San Jose, CA), and Icy software (Quantitative Image Analysis Unit, Institut Pasteur, Paris, France).

Cells in 4-well-type Lab-Tek glass chambers were incubated in 4% paraformaldehyde at room temperature for 20 min, rinsed with PBS, permeated with 0.1% Triton X-100 for 10 min, and then rinsed PBS, 0.1% BSA-PBS(−) (BSA-PBS) and incubated with 3% BSA-PBS for blocking for 1 h. After being washed with 0.1% BSA-PBS, cultured cells were incubated with the anti-TAGE antibody, which was described previously, and dissolved in 1% BSA-PBS at a dilution of 1:100 for 1 h. Cells were then washed three times with 0.1% BSA-PBS and incubated with the HRP-linked goat anti-rabbit IgG antibody (1:100) for 1 h. After washing with 0.1% BSA-PBS (three times) and PBS, cells were incubated with 0.05% DAB and 0.015% H₂O₂ in PBS for 5 min. Thereafter, cells were counterstained briefly with hematoxylin, and observed under the NanoZoomer slide scanner (Hamamatsu Photonics K. K., Hamamatsu, Japan).

We cut 4-μm-thick biopsy tissue sections and mounted them on glass coverslips. Following an in-house optimized protocol, tissues were stained with an antibody against the HLA class II Histocompatibility antigen, DR beta 5 chain (HLA-DRB5 center region) with a rabbit polyclonal antibody (no. OAAB06426, Aviva Systems Biology, CA, US). Normal tonsil tissue was used as a positive control. The stained slides were then scanned on a Hamamatsu NanoZoomer slide scanner and analyzed with NDP viewer software. To estimate the number of cells that stained positive for HLA-DRB5, each stained slide was searched for a hot spot; then, this spot was framed with a 0.75×0.4 mm (0.3 mm²) rectangle; the area included approximately half lamina epithelialis and half lamina propria. We counted all cells in the frame that were distinctly stained with anti-HLA-DRB5 antibodies. The analyzer was blinded to the mucositis grade.

Tissue samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned (5 µm) by the Cell Biology & Bioimaging Core at Pennington Biomedical Research Center. Hematoxylin and eosin-stained paraffin sections of tissue samples were scanned using a Hamamatsu NanoZoomer slide scanner (Hamamatsu, Japan) and viewed through the NDP.view 2 software (https://www.hamamatsu.com/us/en/product/type/U12388-01/index.html).

Liver and kidney specimens from the control and HFCS groups were frozen. Oil Red O was dissolved at a concentration of 6.1 mM in isopropanol and diluted with water to a concentration of 3.7 mM. Tissue sections were washed with water and then incubated with the Oil Red O solution at room temperature for 10 min, followed by the removal of Oil Red O with water, incubation with hematoxylin at room temperature for 5 min, and washing with water. Finally, the sections were coated with malinol mounting medium and observed under a NanoZoomer slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan).