Tissue strips cut from tubular constructs both circumferentially and axially were fixed in 4% paraformaldehyde, embedded in optimal cutting temperature compound (OCT, Tissue-Tek), and frozen. For constructs cultured on porated sleeves, axial strips were taken both from regions along a column of pores and regions between columns of pores. 9 μm thick sections were stained with Lillie’s trichrome. Sections were also stained for collagen I, α1 (Novus, NB600-408). Samples were blocked in 5% normal donkey serum, incubated in primary antibody at a concentration of 1 μg/ml, and stained with Cy3-conjugated anti-rabbit secondary antibody (Jackson Immunoresearch). Hoechst 33342 (Invitrogen, H3570) was used to counterstain nuclei.
The cell distribution was quantified using a custom MATLAB® script.26 (link) Briefly, the user identifies the lumenal tissue surface, and the script determines the distance of the centroid of each cell from the lumenal surface. This quantification was performed on 4X images of Hoechst-stained axial sections. For each section a series of 4X images was pieced together to span the entire length of the axial section.
The cell distribution was quantified using a custom MATLAB® script.26 (link) Briefly, the user identifies the lumenal tissue surface, and the script determines the distance of the centroid of each cell from the lumenal surface. This quantification was performed on 4X images of Hoechst-stained axial sections. For each section a series of 4X images was pieced together to span the entire length of the axial section.
