Excelsior es

For processing samples into paraffin, tissue biopsies from each fibrous capsule were fixed in 4% paraformaldehyde (PFA) for 24 h. Samples were dehydrated in graded series of ethanol and infiltrated with paraffin using a tissue processor (Excelsior ES, Thermo Scientific) and embedded in paraffin. Tissue sections of 6 µm thickness were taken using a microtome (Leica2265, Germany).
For histological analysis, tissue sections were stained with hematoxylin and eosin (H&E) and Masson Trichrome.

The LCSs were harvested after 8 weeks of implantation and they were fixed with 4% formaldehyde. The fixed tissues were rinsed with tap water to remove the fixative for about 2 h. The LCSs were dehydrated in the graded ethanol and cleared in xylene using a tissue processor (Excelsior ES, Thermo Fisher Scientific, USA) and embedded into paraffin blocks sectionally using a paraffin embedding station (EG1150H; Leica, Germany). The paraffin blocks were cut into 5‐μm‐thick sections on a rotary microtome (RM2255; Leica, Germany). The paraffin sections were stained with hematoxylin and eosin (H&E) to identify the tissue structure inside the channels. For immunohistochemistry (IHC) staining of the sections, the following antibodies were used: rabbit polyclonal LYVE‐1 (1:100; NB100‐725, Novus Biologicals, CO, USA) as a lymphatic vessel marker, mouse monoclonal D2‐40 (1:100; ACR266B, Biocare Medical, CA, USA), which is a lymphatic endothelium marker, rabbit polyclonal CD31 (1:2000; ab182981, Abcam, UK), which is an endothelial cell marker. All histologic slides were acquired using a light microscope (Model BX40, Olympus, Japan).

Implants were harvested from the minipigs after 24 weeks and TECs were fixed with 4% PFA, cut into 10 mm × 10 mm cube sections, dehydrated and embedded in paraffin using a tissue processor (Excelsior ES, Thermo Scientific, Waltham, USA). Constructs were horizontally sliced to 5 μm, deparaffinised with Xylene, rehydrated with a decreasing series of ethanol and stained with H & E. Stained slides were scanned with a BIOREVO BZ-9000 microscope (Keyence, Itasca, USA).

Mice liver was collected for histopathological examination. Liver samples were immediately fixed in 10% neutral buffered formalin (NBF) and processed in an auto processor (Excelsior ES, Thermo Scientific, Waltham, MA, USA). After embedding in paraffin, 5-μm sections were stained with hematoxylin and eosin and mounted on glass slides. Digital images were obtained using a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany) at a fixed 200× magnification. The diameter of the portal vein was measured using image measurement software (v 22.1., iSolution DTM, Vancouver, BC, Canada).

MLN4924 was injected in the vitreous of anesthetized 3–4 weeks-old NOD-Scid mice at 0.03, 0.3, 3, 10, 30 µg, and 7 days later mice were sacrificed, the eyes enucleated and incubated in Davidson’s fixative overnight at 4 °C on a shaker. Ethanol dehydration and paraffinization were done with a tissue processor (Excelsior ES, Thermo Scientific, Burlington, ON, Canada). Sections (5 µm) were prepared using an ultramicrotome (Leica Microsystems, Richmond Hill, ON, Canada). Sections were deparaffinised and rehydrated before staining with hematoxylin and eosin (H&E).

B02 was injected in the vitreous of anesthetized 3–4 weeks old Nod-Scid mice at 0.3, 3, 10, 30 µg, and 3 days later the mice were sacrificed, the eyes enucleated and incubated in Davidson’s fixative overnight at 4 °C on a shaker. Ethanol dehydration and paraffinization of the tissue were done in a tissue processor (Excelsior ES, Thermo Scientific, Burlington, ON, Canada). Sections (5 µm) were prepared using an ultramicrotome (Leica Microsystems, Richmond Hill, ON, Canada). Sections were deparaffinised and rehydrated before staining with H&E.

After micro CT scanning, tibial bone specimens were trimmed to five cm length (3 cm defect zone with 1 cm adjacent cortical bone proximally and distally) and fixed in 4% paraformaldehyde for one week before being transferred to 70% ethanol. For histological analysis, the mid-defect regions were sectioned in the transverse and sagittal plane. The sagittally sectioned samples were used for paraffin embedding. For processing decalcified samples into paraffin, bone samples were decalcified in 15% EDTA for 6–8 weeks at 4 °C. The samples were then dehydrated in increasing ethanol concentrations in a tissue processor (Excelsior ES, Thermo Scientific, Waltham, MA, USA), and embedded in paraffin. Five-micrometer sections were obtained using a rotary microtome (RM 2265, Leica, Wetzlar, Germany).
The mineralised bone samples to be cut on the sagittal plane were embedded in methylmethacrylate resin (Technovit 9100, Heraeus Kulzer, Hanau, Germany). Six-micrometer sections of the full length of the defect (5 cm long) were cut using a sledge microtome (Polycut S, Reichert technologies, Depew, NY, USA) and 40–50 µm sections were cut using an EXAKT cutting and grinding system (EXAKT, Norderstedt, Germany). Sections were cut at two thicknesses because the thicker ones preserve gross morphology more accurately and the thin sections reveal more cellular detail.