Aperio versa 200

Confocal images were acquired on an Olympus FV-1000 inverted confocal microscope (Olympus, Waltham, MA). Digital images were collected using the microscope in sequential mode with a line average of 4 and a format of 1024 × 1024 pixels with 8-bit sensitivity. Fluorescent whole-slide images were acquired on the Aperio Versa 200 automated slide scanner (Leica Biosystems, Nussloch, Germany) for consistent, standardized, and objective imaging and quantification of whole slides. BM tissue cores were imaged at 20 × magnification to a resolution of 0.323 μm/pixel. Images were stored on the Digital Image Hub hosted by our institution’s DHSR (Digital Histology Shared Resource). Each whole-slide image was segmented into pieces. The protein expression and co-localization were analyzed using CellProfiler software (Broad Institute, Cambridge, MA) [29 (link),30 (link)]. A CellProfiler pipeline for statistical analysis was developed (described in Supplementary Figure E2, online only, available at www.exphem.org) to evaluate and measure the immunofluorescence signals, containing the number, shape, size, and median intensity of signals (RIPK1, pMLKL, cleaved caspase-3, CD34, CD71). Analysis of each slide included at least 5,000 cells.

Rat femurs were fixed in 10% NBF and decalcified with Cal-Ex^® II (Fisher Chemical CS5114D Cal-Ex^® II Fixative/Decalcifier). Following decalcification and fixation, samples were dehydrated in increasing concentrations of alcohol (70%, 95%, 100%), followed by xylene before embedding in paraffin and cut into 5 micron slices (Accu-Cut SRM 200 Rotary Microtomoe, Sakura Finetek USA, Torrance, CA, USA). Slides were stained with Hematoxylin and Eosin, Goldner’s Trichrome, and Safranin-O/Fast-green. Images were obtained with Aperio VERSA200 (Leica Biosystems, Inc., Buffalo Grove, IL, USA) with a 40X HC PL APO objective with .95 numerical aperture and captured with Aperio ImageScope v12.4.3 (Leica Biosystems, Inc., Buffalo Grove, IL, USA).

To detect apoptotic cells in the burn tissue, histologic cross-sections were stained using a Click-iT^® Plus TUNEL Assay kit (Life Technologies, Carlsbad, CA) following the manufacturer’s directions. Briefly, tissue sections on slides were deparaffinized with xylene and re-hydrated with an ethanol series before fixation in 4% paraformaldehyde for 15 minutes. The tissue was permeabilized with Proteinase K for 15 minutes and additionally fixed with 4% paraformaldehyde for 5 minutes. The tissue was incubated in TdT Reaction Buffer for 10 minutes before addition of the TdT Reaction Mixture for 60 minutes at 37°C. Tissue sections were then washed with 3% bovine serum albumin (BSA) in 0.1% Triton X-100 for 5 minutes, rinsed with PBS, and incubated with Click-iT Plus TUNEL reaction cocktail for 30 minutes at 37°C in the dark before washing with 3% BSA in PBS for 5 minutes. The samples received a final rinse in PBS before being cover slipped using ProLong Gold Antifade Mountant with 4ʹ,6-diamidino-2-phenylindole (DAPI; Life Technologies). Fluorescent images were acquired using a ×10 objective and Ex/Em wavelengths of 590/615 nm for Alexa Fluor 594 to show damaged DNA strands and 358/461 nm for DAPI as a general nuclei stain on a Leica Aperio Versa 200 (Leica Biosystems, Inc., Buffalo Grove, IL) slide scanner.