Leica rm2255

Images of hematoxylin and eosin-stained slides were created on an Aperio ScanScope XT (Leica Biosystems, Buffalo Grove, Ill). The Aurora mScope viewer (Aurora Interactive Ltd, Montreal, Quebec, Canada) was used to view the images and obtain images at × 20 magnification. A pathologist examined a digitized hematoxylin and eosin-stained slide to determine and outline tumor nests, directing the laser capture microdissection (LCM) to areas of high tumor cell content on the adjacent FFPE sections. A total of 5 to 10 sections (10-μm thick) from each FFPE block were cut on a microtome (Leica RM 2255; Leica Biosystems), air-dried for ≥ 2 hours, and placed at 60°Cfor 30 minutes. The sections were stored at 4°C until use or were immediately stained with a rapid hematoxylin and eosin ethanol-based staining protocol. Within 10 minutes of air-drying, the slide was placed on the LCM stage of the Arcturus PixCell instrument (Thermo Fisher Scientific, Waltham, Mass) for microdissection. The desired cells were microdissected into the cap of a 500-μL, safe-lock tube filled with 50 μL of ALT buffer (Qiagen, Valencia, Calif). This procedure was repeated on the next slide until a total of 2000 to 5000 cells of interest had been captured for each case.

Native and decellularized tissues were fixed in 10% phosphate-buffered formalin, serially dehydrated, embedded in paraffin, and then sectioned (6 μm thickness) with a microtome (Leica RM2255, Leica Biosystems, Buffalo Grove, IL, USA). Samples were rehydrated and stained with haematoxylin & eosin (H&E, Sigma-Aldrich) or 4′,6-diamidino-2-phenylindole, dilactate (DAPI, Life Technologies, Carlsbad, CA, USA). H&E-stained samples were imaged using an Olympus SZX16 stereo microscope while DAPI-stained sections were examined with an Olympus CKX41 inverted microscope using fluorescent excitation at 405 nm.