Brief Note dissected were placed in Hank’s balanced salt solution without Ca++ and Mg++ (HBSS) (Sigma-Aldrich, St. Louis, MO, USA), and fixed for 15 min at room temperature with 4% formaldehyde prepared from paraformaldehyde in phosphate-buffered saline (PBS). The aortae were dehydrated in graded ethanol and embed- ded in paraffin. Paraffin sections (5 μm thick) were mounted on silanized slides (Dako, Inc., Carpinteria, Ca, USA). A total of two aortae for each stage obtained from three different lots of fertilized chicken eggs were processed. Indirect immunofluorescence Serial deparaffinized cross sections were air dried and equilibrated in PBS for 10 min. Non- specific antibody staining was blocked by incu- bating sections in PBS containing 2% bovine serum albumin (BSA) for 30 min at room tem- perature. Sections were incubated overnight at 4ºC in a humid chamber with mouse mono- clonal anti-human endocan/ESM-1 antibody (clone MEP08) (Lunginnov, Lille, France) or mouse monoclonal antibody anti α-SM actin (clone 1A4) (Sigma-Aldrich). After the sec- tions were washed several times in PBS, anti- mouse FITC-conjugated secondary antibody (Dako) was applied for 30 min. The sections were again washed in PBS, and coverslipped with mounting medium (IMMU-mount; Shandon, Pittsburg, PA, USA). Negative con- trols were produced by the use of purified nor- mal serum or PBS in place of primary antibody. A IX81 Olympus inverted microscope with the Fluo-View confocal laser scanning configura- tion (CLSM) (Olympus America, Inc., Melville, NY, USA) was used to examine the sections. Fluorescence intensity was measured by using a processing software program (FV10. ASW version 02.01.01.04, Olympus Corporation). Cell cultures Aortae from days E11-E12 (stages 37 and 38) were dissected in HBSS (Sigma-Aldrich) at 37ºC. Segments, approximately 8 mm2 in sur- face area, were isolated (distal to the aortic arches) and opened along longitudinal axis. Explants were rinsed in HBSS (Sigma-Aldrich) and left in medium 199 with Earle’s salts with L-Glutamine (Sigma-Aldrich) containing 0.025% BSA (Sigma-Aldrich), 100 μg/mL strep- tomycin, and 100 U/mL penicillin (GIBCO, Invitrogen, Carlsbad, CA, USA) for 5-10 min before starting the assays. For simplicity, this medium is referred to as serum-free medium (SFM). Aortic explants were placed with the endothelial apical side down on fibronectin- coated 35 mm Petri dishes (Nunclon, Delta, IL, USA) containing 300 μL of SFM. They were incubated at 37ºC in a humidified atmosphere consisting of 5% C02 and 95% air for 4 h. After this interval, 1 mL of SFM was gently added to each dish. Two h later, the adhered explants were removed with the aid of a thin needle, leaving a monolayer of retracted endothelial cells that exhibited zones denuded of cells or wounds. Monolayers were examined with an inverted microscope (IX-70 Olympus, Olympus America Inc.). Cell cultures were initiated when the monolayers of endothelial cells were rinsed 5 times with HBSS and incubated for 2 h at 37ºC with 5% CO2. At the end of this time, the medium of the monolayers was switched to SFM supplemented with recombinant epider- mal growth factor (EGF) (R&D Systems, Inc., Minneapolis, MN, USA) (100 ηg/mL) and 0.1% chicken serum (ChS) (GIBCO) and incubated for an additional 10-12 h period. Indirect immunofluorescence Fixed and permeabilized cells were processed for immunostaining as described above using the following antibodies: a mouse monoclonal anti-human endocan/ESM-1 (clone MEP08) (Lunginnov), a rabbit polyclon- al anti-human von Willebrand´s factor (vWf) (Dako), a mouse monoclonal antibody raised against native chick brain microtubules (clone DM1A) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), a mouse monoclonal antibody anti-human β3-integrin (GPIIIa, CD61) (clone 25E11) (Millipore Chemicon Corporation, Billerica, MA, USA), and a rabbit polyclonal anti-phospho-FAK (Tyr-397) (Sigma-Aldrich). Negative controls were performed by omitting the primary antibody incubation step or by using non-immune serum instead of primary antibody. A confocal laser scanning microscope (CLSM) (Eclipse TE-300 Nikon inverted micro- scope) (Nikon Instruments Inc., Melville, NY, USA) equipped with a Nikon objective Plan- Apo BC x60, 1.2 wi coupled to a C1-LU2 unit Argon cooled air (488 ηm) laser was used to examine the cultures. The laser unit was con- trolled by a D-eclipse C1 interface. Figure 1. Overlay of transmission and red or green fluorescence images of serial paraffin cross sections of chicken embryo aorta at day E12 of development immunolabeled with either anti-αSM actin or anti-endocan antibodies. In this stage, it is possible to distin- guish mesenchymal cells (mc) adjacent to the endothelium (e) forming an intimal thick- ening (IT). Strong α-SM actin immunoreactivity is detected in few mesenchymal cells (mc) and in the cells arranged in lamellar layers (lc) but not in interlamellar cell layers (ilc). Strong endocan immunoreactivity is detected in the endothelium (e) as well as in some adjacent mesenchymal cells (mc). Histograms represent the percentage of fluores- cence intensity for α-SM actin (red) and endocan (green) after setting the ROI on the superimposed images. [page 74] [European Journal of Histochemistry 2011; 55:e13]