Whole embryos were imaged using a Nikon NZ100 dissecting microscope, and sections were imaged in an Olympus BX61 fluorescence compound microscope.
Using optical microscope, the sections were examined for evaluation of bone formation and integration of the reconstructed areas into the neighboring bone tissue.
For examination under scanning electron microscope, duodenum sections were fixed using 2.5%% glutaraldehyde in phosphate buffer saline (PBS) (pH 7.4).
After sectioning and adherence to gelatin-coated microscope slides, sections were sequentially immunostained in a TedPella Biowave microwave and antibody dilutions used in this experiment are detailed in Supplemental Table 1.
The slides were visualized under light microscope, entire tissue sections were examined, and three random regions were selected and photographed using a digital camera.
Imaging of whole mount labeling was with a Zeiss LSM510 confocal microscope, individual optical sections were flattened for each image, except for Fig. 2B,D, which show single optical sections.
Immunofluorescence microscopy was performed with either a Leica DM-RA2 microscope, or a Leica TCS SP5 confocal laser-scanning microscope and confocal optical sections were created using Leica confocal software.
Analysis was performed under Olympus BX 51 microscope, and pictures of the sections were taken by a microscope digital camera system (DP50, Olympus, Japan).
They were mounted in 80% glyercol on the depression slides and imagining was performed using Olympus Fluoview 1000 confocal microscope: serial Z-stack sections were taken with 0.5 µm step size.
Fluorescence confocal microscope images of the sections were obtained, as described earlier.
