Constructs were collected and fixed in 4% paraformaldehyde. IF was performed as previously described (Zieske et al. 2001 (link)). Following fixation, the constructs were incubated at 4°C overnight with the primary antibody—anti-type III collagen (1:40: Southern Biotech; Birmingham, AL), or anti-SMA (1:50: Dako North America; Carpinteria, CA)—diluted in 1%BSA + 0.1%Triton-X. The constructs were then washed and incubated overnight at 4°C with the corresponding secondary antibody—donkey anti-goat IgG (1:200, type III collagen), or donkey anti-mouse IgG (1:200, SMA)—diluted in 1%BSA + 0.1%Triton-X. Phalloidin-rhodamine (Invitrogen; Carlsbad, CA), which binds to the f-actin in all cells, was also used. Constructs were counterstained with TOPRO-3 iodide (1:1000, Invitrogen), a marker of all cell nuclei. Negative controls, where the primary antibody was omitted, were run with all experiments. Constructs were washed, mounted with Vectashield Mounting Media (Vector Laboratories; Burlingame, CA), observed and photographed using a confocal TCS-SP2 Leica microscope (Leica Microsystems; Bannockburn, IL). In addition, construct thicknesses were also measured with the confocal microscope, beginning with the first cell visible at the top of the construct and the last cell visible at the bottom. Data was averaged and analyzed.