mouse anti-human antibody in a concentration of 8 μg per ml as

primary antibody and donkey anti-human Alexa Fluor 488 at the

dilution of 1:500 as secondary antibody.

1. Block the dedicated graft sample in PBS containing 3% donkey

serum

for

45

min

at

room

temperature

in

a

2

ml

microreaction tube.

2. Transfer sample in a 2 ml microreaction tube containing an

excess of primary antibody solution to ensure that the sample is

completely immersed.

3. Incubate over night at 4
C.

4. Wash samples three times with PBS by transferring to 2 ml

eppendorf tubes filled with PBS.

5. Incubate the section in a 2 ml microreaction tube containing an

excess of secondary antibody solution with one drop per ml

Nucblue reagent to ensure that the sample is completely

immersed and incubate for 1 h in the dark at room

temperature.

6. Wash three times with PBS.

7. Cut the circular sample longitudinally and spread the luminal,

colonized surface flat on a microscope slide.

8. Acquire images in accordance to the specifications as described

in the manufacturers manual or data sheet.

4

Notes

1. Our bioreactor chamber was customized using a 3D printer. It

features four barb connectors on the inside and six on the

outside. Two grafts could be perfused simultaneously.

2. The glass flask used as medium reservoir was connected to the

perfusion circuit through two of the three outlets in the flask.

Additionally, for sterile gas exchange, a 22 μm gas filter was

connected to the reservoir. One inlet in the bottle cab was used

to acquire media for substrate balancing. Another one was

connected with a silicon tube to the bioreactor chamber in

order to guarantee pressure release.

3. Make two loops around the tubes over the barb connectors and

secure the loose ends with a surgical knot on top of the tube.

Make sure, to avoid making air knots.

4. If you chose to run controls under 2D conditions for your

experiments, the growth kinetics of your respective cell line

should be tested in advance to ensure that the cell number

normalized to surface area similar to the tested grafts does

allow for cell growth without reaching high confluency levels

In Vitro Colonization of Vascular Grafts

219