Permount mounting medium

Luxol fast blue (LFB) staining was performed according to the manufacturer’s instructions (Abcam, UK). Briefly, spinal cord sections were rehydrated and incubated in 0.1% Luxol fast blue solution at 40 ^oC overnight. Excess stain was rinsed with 95% ethanol. Slides were washed in distilled water, immersed in 0.05% lithium carbonate solution for 30s, and then dehydrated in serial ethanols and mounted in Permount mounting medium (Sigma-Aldrich, USA). In addition, selected sections were stained with hematoxylin and eosin (H&E) using standard procedure.

Formalin-fixed, paraffin-embedded (FFPE) tumor, lung, and liver sections were de-paraffinized, rehydrated in Sub-X (Leica, Buffalo Grove, IL, USA) and graded solutions of ethanol, and stained with hematoxylin and eosin. FFPE tumor sections were de-paraffinized, rehydrated in Sub-X and graded solutions of ethanol, quenched with 0.3 % H₂O₂ (Sigma), rinsed with Tris-NaCl-Tween buffer (TNT), which consisted of 0.1 M Tris-HCl (pH 7.5; Sigma), 0.15M NaCl (Sigma), and 0.05 % Tween-20 (Invitrogen). Tumor sections were then blocked with 1 % BSA, and stained with primary antibodies obtained from Abcam against GFP, dsRed, SERPINE1, or matrix metalloproteinase-2 (MMP-2) overnight at 4 °C. Each tumor section was subsequently washed in TNT buffer. Tissue sections were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam) for 1 hour at room temperature, and washed with TNT buffer. For colorimetric staining, slides were then incubated in 3,3′-Diaminobenzidine (DAB; Vector Laboratories), washed with TNT, counterstained with hematoxylin (Thermo Scientific), and rinsed with deionized water. Slides were dehydrated in graded solutions of ethanol, followed by SubX in the final step, and sealed with Permount Mounting Medium (Sigma). After staining, images were acquired at × 10 and × 40 magnification with the ScanScope CS2 (Aperio, Vista, CA, USA).

Fixed stem segments were dehydrated in an automated tissue processor (Leica Microsystems Inc., Buffalo Grove, IL) following the manufacturer’s instructions. The samples were then embedded with Paraffin Plus (Fisher Scientific Co., Houston, TX) using a Leica embedding machine (Leica Microsystems Inc., Buffalo Grove, IL). Longitudinal and transverse sections of the cankers were made using a rotary microtome (Leica Microsystems Inc., Buffalo Grove, IL) set to a thickness of 20 µm. Three segments were sectioned transversely through the top, middle, and bottom of each canker and the remaining three were sectioned longitudinally through the center of each canker. Several sections were made at each location. All sections were placed on microscope slides (Fisher Scientific Co., Houston, TX), de-waxed in Histo-Clear (Fisher Scientific Co., Houston, TX), stained using a Safranin-O Fast-green protocol [27] , and mounted using Permount mounting medium (Sigma-Aldrich Co., St. Louis, MO). Sections were examined by light and fluorescence microscopy using a Zeiss Axio Imager M2 microscope. Blue auto-fluorescence viewed with ultraviolet light (Excitation filter G 365, Beam Splitter FT 395, Emission filter BP 445/50) and green auto-fluorescence viewed with blue-green light (Excitation filter BP 450–490, Beam Splitter FT 510, Emission filter BP 515–565) were used to visualize host responses.

Formalin fixed calvaria were decalcified in 10 % EDTA (Sigma), paraffin embedded, and sectioned. Tissue sections stained with hematoxylin and eosin (Richard-Allan Scientific, Thermo Scientific; Waltham, MA), Aniline Blue (Sigma), or Tartrate-Resistant Acid Phosphate (TRAP; Sigma). After staining, slides were dehydrated in graded solutions of ethanol and Sub-X in the final step, and sealed with Permount Mounting Medium (Sigma). Images were acquired and quantified with the ScanScope CS2 (Aperio, Vista, CA) running Image Scope (Aperio).