First 11 pages of thesis. - OPUS - Universität Würzburg

along with an ÄKTA HPLC system (Amersham Biosciences, GE Healthcare).
The mobile phase was composed of a gradient containing 2.6 column volumes
of 37- 47% acetonitrile containing 0.1% trifluoroacetic acid. Oxyethidium was
quantified with a fluorescence detector (Jasco, UK) at 580 nm (emission) and
480 nm (excitation). The detected oxyethidium was normalised to the sample’s
protein content 199 .
2.2.6 Tissue preparation and immunohistochemistry
Aortic arches were cleaned of adherent tissue, embedded in Tissue-Tek ®
(Sakura Finetek) and snap-frozen in liquid nitrogen. Serial sections (5 µm) were
cut at -20°C, mounted on silane treated slides (SuperFrost ® Plus, Menzel GmbH
& Co KG, Braunschweig, Germany), thoroughly air-dried and fixed in acetone
for 10 minutes at room temperature prior to staining. The slides were allowed to
dry at room temperature for 1 hour followed by washing thrice with PBS. The
sections were incubated with 0.3% hydrogen peroxide in methanol for 30
minutes and washed with PBS. A commercially available blocking reagent
(Dako REAL, antibody dilutent) was used for 30 minutes and subsequently
incubated along with the primary anti nitrotyrosine antibody (1:80 dilution,
Upstate Biotechnology, Inc.) for 1 hour. After washing off unbound antibody, the
sections were incubated for 30 minutes with the secondary biotinylated anti
rabbit IgG antibody (1:200 dilution, Vector Laboratories, Inc.) at room
temperature. Following three PBS washing steps sections were stained for 30
minutes with ABC reagent (Vectastain ® ABC kit, Vector Laboratories, Inc.).
Sections were then washed with PBS and incubated with DAB reagent for 5-20
- 53 -