Whole-mount Intestine Clearing and Imaging

Foxl1 Cre; Rosa-mTmG and C57BL/6 adult mice were anesthetized, intestine was dissected and fixed in 4%PFA for 24hr at 4°C. Following washes with PBS for another 24hr at 4°C, the intestine was incubated in Hydrogel solution for 24hr at 4°C ^{27 (link)}. After 3hr at 37°C, the hydrogel-embedded intestine was placed in an X-CLARITY^TM ETC chamber (LOGOS biosystems) for electrophoretic tissue clearing for 7hr. The cleared intestine was immunostained with goat anti-GFP (1:100; Abcam) or PDGFRα (1:100; R&D) and APC-conjugated anti-mouse EpCAM (1:100 Biolegend) or rabbit anti-EpCAM (1:100; Abcam). For Foxl1 antibody staining, following clearing an antigen retrieval in citrate buffer was performed (as described above). Primary and secondary antibodies were incubated for 48hr at 4°C each. The stained intestine was placed en block on an image slide using 1mm depth adhesive silicone isolator, mounted in X-CLARITY^TM mounting solution and imaged using confocal scanning. Z-stacks projections were compiled using Volocity software.