St5010

After fixation, strips were stepwise dehydrated (Leica TP1020, Semi-enclosed benchtop tissue processor, Germany) and embedded in paraffin wax blocks. Strips were either embedded to get 1) axial cross-sections – such that the lumen was oriented perpendicular to the face of the wax block or 2) with the luminal side of the strip flush with the face of the wax block to achieve cross-sections radially through the wall thickness. The integrity of the strip after mechanical testing dictated which of these options was most feasible; specifically, if it was not possible to orient the samples such that axial cross-sections could be obtained, the samples were oriented to get radial cross-sections. Samples were sectioned at 7 μm using Feather C35 microtome blades. Samples were stained with Haematoxylin & Eosin (H&E), picrosirius red (PSR) and Verhoeff’s elastin (Leica ST5010, Autostainer XL, Germany). Brightfield imaging of all stains was performed on an Aperio CS2 microscope with ImageScope software V12.3 (Leica Biosystems Imaging, Inc., Vista, California). Polarised light microscopy (PLM) was performed on the PSR-stained samples using an Olympus BX41 microscope with Ocular V2.0 software (Teledyne Photometrics, Tuscon, Arizona). Two PLM images were taken for each slice, 45° to each other, with the exposure time kept consistent across all samples.

At 4 weeks after surgical operation (postop), animals were humanely killed under deep anesthesia. Samples were extracted and fixed in 4% paraformaldehyde. After decalcification, the specimens were embedded in paraffin and sectioned into 4 μm thick sections. For morphological study, sections were stained with hematoxylin and eosin (HE) with an autostainer (ST5010, Leica, Germany). For immunohistochemistry analysis, sections were subjected to immunostaining with mouse monoclonal anti-GFP (1 : 100, Santa Cruz, USA), rabbit polyclonal anti-rat osteocalcin (OCN) (1 : 100, Santa Cruz, USA), and rabbit polyclonal anti-human OPN antibodies (1 : 100, Proteintech Group, USA). Then all samples were photographed.

The whole brain was carefully removed at 4 °C and fixed with 4% polyformaldehyde for 24 h. The brain was dehydrated with ethanol, cleared with anhydrous ethanol xylene and embedded in paraffin, then cut into 4 μm sections by paraffin microtome (Leica RM2245, Witzler, Germany). After it was dried, slices were used for HE staining with an automatic staining machine (Leica ST5010, Witzler, Germany). Finally, pathological analysis of the hippocampus was performed by a digital slice scanning system (3 DHISTECH MIDI, Budapest, Hungary).

The samples were washed with BBS distilled water and transferred to 70% ethyl alcohol for long-term storage. Afterwards, the colon tissues were embedded in paraffin and sectioned at five μm thickness (Leica, RM2245). Hematoxylin and 0.5% eosin were used for histopathological staining (Leica, ST5010). Under a light microscope (Leica, DM2000), tissue sections were viewed at a magnifying power of ^*100. Each group underwent four biological verifications.