Nanozoomer rs

Colorectal cancer tissues were collected in the Department of Frontier Surgery, Chiba University Hospital, as previously described8 (link). Paraffin-embedded blocks were cut into 2.5-μm-thick sections using the microtome, REM-710 (Yamato, Saitama, Japan). Immunostaining was performed with the diaminobenzidine (DAB) chromogen (EnVision + Kit/HRP; Dako, Glostrup, Denmark) and nuclei were stained with Mayer’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan). Stained sections were scanned on a NanoZoomer RS digital slide imaging system (Hamamatsu Photonics, Hamamatsu, Japan). Tissues used for immunofluorescence were embedded in OCT compound (Sakura Finetek, Tokyo, Japan), snap-frozen in liquid nitrogen, and cut into 5-μm-thick sections using a cryostat (Hyrax C50; Carl Zeiss, Jena, Germany). The procedure for the immunofluorescence of tissues was described below. The protocol for the collection and use of tissue samples was approved by the Ethics Committees of the Graduate School of Medicine, Chiba University and the Proteome Research Center, National Institutes of Biomedical Innovation, Health, and Nutrition. Written informed consent was obtained from each patient before surgery.

Astrocytes, HMC3 and U87MG were labeled with CellTrackers® as previously described before seeding. 3D GBM spheroids were cultured for 3 days before being washed with DPBS, fixed with 4 v/v% formaldehyde, washed again twice with DPBS and embedded into Cryomatrix™ before being snap-frozen using isopentane. Embedded and frozen 3D GBM spheroids were cut into 6 μm thick cryosections, air-dried, rehydrated with PBS and immediately mounted with Fluoroshield™ with DAPI (Sigma Aldrich) before being imaged using Nanozoomer-RS (Hamamatsu Photonics, Hamamatsu, Japan). Scanned images were analyzed using Image J (Public, developed by Wayne Rasband (NIH)).

Double immunohistochemical staining with mouse anti-CD34 (Dako, Glostrup, Denmark) and anti-SMA (Dako) was performed to identify endothelial cells and smooth muscle cells of the arterial media (Figure 1A). The PAS stain was then applied to the sections to identify the tubular basement membranes, Bowman’s capsules, sclerotic glomeruli, and interstitial fibrosis (Figure 2F,G, Figure 3A,G, Figure 4F,G, Figure 5C,F,G and Figure 6G). Details of the tissue preparation are described elsewhere [7 (link),8 (link),9 (link),10 (link)]. Each stained specimen was digitized using virtual slide microscopy (NanoZoomer RS™; Hamamatsu Photonics, Hamamatsu, Japan), using a 40× optical lens.