solution should be prepared in an excess of volume to ensure

that the sample is completely immersed. We used 1.0 ml of

staining solution in a 2 ml microreaction tube for 1 cm of

sample (see Note 9).

2. Incubate the dedicated sample for 45 min at room temperature

in the dark.

3. Wash three times with PBS (see Note 10).

4. Cut open the circular sample in longitudinal orientation and

spread the colonized luminal surface flat onto a microscope

slide.

5. Acquire images according to specifications as described in the

manufacturers’ manual.

Exemplary images are shown in Fig. 3.

3.10

Acridine Orange

Staining

A quick and easy option to get a visual impression of cell-survival,

cell-morphology and its distribution on the artificial scaffold can be

achieved by Acridine Orange Staining, a fluorescent dye that

reports both, cytoplasmatic RNA as well as DNA in the nuceli

[18, 23].

1. Prepare the staining solution freshly (0.1% acridine orange

supplied stock solution in PBS). The staining solution should

be prepared in an excess of volume to ensure that the sample is

completely immersed. We used 1.0 ml of staining solution in a

2 ml microreaction tube for 1 cm of sample.

2. Place the section in a 2 ml microreaction tube and incubate for

45 min at room temperature in the dark.

3. Wash three times with PBS.

4. Cut open the section and spread it out flat on a microscope

slide.

5. Acquire images according to specifications as described in the

manufacturers manual or data sheet.

Exemplary images are shown in Fig. 3.

3.11

CD31

Immunofluorescence

Staining

Complementary to the cytoskeleton staining as described in Sub-

heading 3.9, staining of the samples for endothelial cell-cell junc-

tions allows investigating the cell-lining in formed colonies and

layers. For this purpose, immune staining of CD31 or PECAM-1,

which is expressed on endothelial cell-cell junctions and involved in

platelet and leukocyte transmigration, thus also representing a

marker for endothelial functionality, is widely used and accepted

in literature [24, 25].

Prepare the antibody dilutions of primary and secondary anti-

bodies with concentrations recommended by the manufacturer in

PBS with 3% donkey serum in advance. We used CD31/PECAM-1

218

Max Wacker et al.