Intestinal and liver samples were fixed overnight in paraformaldehyde, l -Lysine pH 7.4 and NaIO4 (PLP buffer). They were then dehydrated in 20% sucrose for at least 4 h and included in OCT compound (Sakura). Alternatively, whole pieces of tissue, were stained directly after fixation in PLP to image whole mount. 10 μm cryosections were rehydrated, blocked with 0.1 M Tris-HCl pH 7.4, 2% FBS, 0.3% Triton X-100 and stained with antibodies recognizing the following proteins: PV1 (clone MECA32, BD Pharmingen, 1:100), ZO-1 (clone ZO1-1A12, Invitrogen, 1:100), CD34 (clone RAM34, eBioscience, 1:50).
Tissues were then incubated with the appropriate fluorophore-conjugated secondary antibody. Before imaging, nuclei were counterstained with 4′,6-diamidin -2-fenilindolo (DAPI). Confocal microscopy was performed on a Leica SP8, using oil immersion objectives with x40 or x63 magnification. Fiji (ImageJ) software package was used for image analysis and fluorescence quantification.
Tissues were then incubated with the appropriate fluorophore-conjugated secondary antibody. Before imaging, nuclei were counterstained with 4′,6-diamidin -2-fenilindolo (DAPI). Confocal microscopy was performed on a Leica SP8, using oil immersion objectives with x40 or x63 magnification. Fiji (ImageJ) software package was used for image analysis and fluorescence quantification.