Immunohistology

2.2.1 PECAM-1 (CD31)

Endothelial cells were demonstrated in frozen sections by staining for CD31 (PECAM-1). PECAM-1, an endothelial cell adhesion molecule, is a widely used panendothelial cell marker, and facilitates the assessment of the vascular status of a tissue (NEWMAN 1997, EBERHARD et al. 2000). Frozen sections were fixed in

acetone for 10 min, dried and then washed in Tris-buffered saline (TBS, pH 7,4).

After pre-incubation with 10% normal goat serum in TBS, slides were incubated with a monoclonal rat antibody raised against the murine homologue of CD31 (Pharmingen, San Diego, CA, USA). This antibody was used in a dilution of 1:2000 in TBS, and 2% normal goat serum were added. After over-night incubation, slides were washed and treated with a polyclonal goat antibody, directed against rat IgG (Dianova GmbH, Hamburg, Germany) in a concentration of 1:200 in TBS. 2% normal goat serum and 4% normal mouse serum were added.

For light microscopical detection, a biotinylated antibody was used. This was followed by the ABC-detection system. The latter was either conjugated to alkaline phosphatase or to peroxidase (Vector, Burlingame, CA, USA). According to the employed enzyme, Vectastain AP substrate kit I or DAB (Vector, Burlingame, CA, USA) were used as substrates. Slides were counterstained with hematoxylin, dehydrated and permanently mounted.

For immunofluorescent detection, a fluorescein isothiocyanate (FITC)– or Cy3-conjugated secondary antibody was used. Slides were then stained with DAPI, a cell nuclei marker, and mounted in Fluoromount (Southern Biotechnology Associates, Bermingham, AL, USA). Sections without the primary antibody and sections with an antibody to an irrelevant antigen served as negative controls.

2.2.2 Ki-67 / PECAM-1 double staining

Sections were treated as described above to stain for PECAM-1. After incubation with the secondary antibody, conjugated to FITC, sections were pre-treated again with 10% normal goat serum in TBS and were finally incubated with a polyclonal rabbit antibody, raised against the murine Ki-67 protein (Dianova GmbH, Hamburg, Germany). This antibody was diluted 1:100 in TBS and 2% normal goat serum were added. After over-night incubation, setions were treated with a Cy3-conjugated goat antibody, raised against rabbit IgG (Dianova GmbH, Hamburg, Germany), diluted 1:200 in TBS with 2% normal goat serum and 4% normal mouse serum. Double immunoreactive cells were identified as cells that revealed a red intranuclear staining

(Ki-67) which is surrounded by a green cytoplasmic staining (CD31), resulting in a small yellow-appearing zone around the nucleus, where both colors superimpose.

Sections without either primary antibody served as negative controls.

2.2.3 Vascular endothelial growth factor (VEGF)

For detection of VEGF-immunoreactivity, paraffin sections were deparaffinized in xylene, rehydrated in graded alcohols and washed in 0.05 M TBS (pH 7.4). They were then incubated for 15 min in 3% H2O2 to quench endogenous peroxidase activity and subsequently incubated with normal rabbit serum (10% in TBS) to block unspecific binding of the secondary antibody. Sections were incubated over-night with an affinity-purified goat polyclonal antibody raised against a peptide mapping at the amino terminus of the mature form of murine VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) in a dilution of 1:150 in TBS. After washing in TBS, slides were incubated with a biotin-conjugated rabbit anti-goat IgG antibody (Dianova GmbH, Hamburg, Germany) diluted 1:300 in TBS for 30 min at room temperature. After thorough washing, slides were incubated with a PAP (goat) complex (DAKO, Hamburg, Germany), washed, and then incubated with a peroxidase-conjugated avidin-biotin-complex (Vector, Burlingame, CA) for 30 min at room temperature each.

Labeling with peroxidase was developed using a 3`3-diaminobenzidine substrate kit (Vector, Burlingame, CA), counterstained with methylen green (DAKO, Hamburg, Germany), dehydrated and mounted. For negative controls, the primary antibody was incubated with a five-fold excess of the blocking peptide (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight.

2.2.4 Angiopoietin-1 and Angiopoietin-2

For the detection of Ang-1 and Ang-2 –immunoreactivity in paraffin sections of mouse skin samples, a similar protocol was used as has been described for VEGF (2.2.3). As primary antibody, an affinity-purified goat polyclonal antibody raised 1) against a peptide mapping at the amino terminus of human angiopoietin-1 or 2)

against a peptide mapping at the carboxy terminus of human angiopoietin-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used in a dilution of 1:100 and 1:200, respectively. Both antibodies were known to react with the equivalent protein of mouse origin.

2.2.5 VEGFR-1 (Flt-1) / PECAM-1 – double staining

Double-staining for VEGFR-1 and PECAM-1 was performed on frozen sections as follows: Endogenous peroxidase was quenched in 3% H2O2 for 15 min. Endogenous avidin and biotin were blocked using an avidin/ biotin blocking kit (Vector, Burlingame, CA) according to the manufacturer′s protocol. All steps were interspersed by washing in TBS. Sections were pretreated with normal goat serum and incubated with a rat anti PECAM-1 antibody as described above. After washing, sections were pretreated with TNB buffer (NEN Life Science Products, Boston, MA) for 20 min at room temperature and incubated over-night with a rabbit affinity-purified polyclonal antibody raised against a peptide mapping at the carboxy terminus of Flt-1 of human origin (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000 in TNB buffer. Sections were washed in TNT (NEN Life Science Products, Boston, MA) and incubated with a biotin-labeled anti rabbit-IgG antibody raised in goat (Dianova, Hamburg, Germany), diluted 1:200 in TNB. After washing in TNT, sections were incubated with a streptavidin-horseradish peroxidase complex (NEN Life Science Products, Boston, MA), diluted 1:100 in TNB buffer and with a tyramide-fluorescein complex (NEN Life Science Products, Boston, MA), diluted 1:50 in amplification diluent (NEN Life Science Products, Boston, MA). Sections were washed in TNT and TBS and were treated with a goat antibody against rat IgG, labeled with Cy3 (Dianova, Hamburg, Germany). Sections were washed in TBS again and mounted in Fluoromount (Southern Biotechnology Associates, Birmingham, AL).