CVR-2011-414R1 eNOS & EPC mediated neoangiogenesis
Supplementary Data
Supplementary Methods
Immunofluorescence analysis
For pre-treatment slices with 3 µm thick deparaffinised sections were placed in Coplin jars
with 0.05% citraconic anhydride solution, pH 7.4 for 1 hour at +98°C and then incubated
overnight at 4°C with the first antibody, followed by the appropriate secondary antibody at
37°C for 1 hour. Immunofluorescence studies were performed by applying polyclonal
antibody against podocalyxin (R&D Systems, USA) to detect capillaries and endothelial cells.
Monoclonal antibody against α –sarcomeric actin (clone5c5, Sigma-Aldrich, USA) was used
to detect cardiomyocytes. In co-immunostainings of bone marrow transplanted mice for
podocalyxin a GFP-specific antibody (abcam, United Kingdom) was used to enhance GFPfluorescence.
Cycling cardiac cells were identified by immunostaining for proliferation
marker Ki67 (Novocastra, United Kingdom). Proliferating endothelial cells and fibroblasts
were identified by co-immunostaining for podocalyxin and fibronectin. Monoclonal antibody
against fibronectin (abcam, United Kingdom) was used. To detect eNOS-positive cells in
bone marrow of BMT-mice a polyclonal antibody against eNOS (abcam, United Kingdom)
was applied. FITC-, TRITC–, Biotin- and Peroxidase-conjugated anti-mouse IgM, anti-mouse
IgG, anti-rabbit IgG, and anti-goat IgG (all Dianova, Germany) were used as secondary
antibodies and for amplification if necessary. To perform wash steps 4xSSC buffer was used.
Sections were counterstained with DAPI (Calbiochem, Germany) and mounted with
fluorescent mounting medium (Vectashield, Vector Laboratories, Burlingame, CA) for
fluorescence microscopic analysis. All sections were evaluated using a Nikon E600
epifluorescence microscope (Nikon, Germany) with appropriate filters. In addition
1
CVR-2011-414R1 eNOS & EPC mediated neoangiogenesis
colocalization of stainings (e.g. podocalyxin and GFP) were controlled using a confocal
microscopy unit (QLC100, VisiTech; United Kingdom on a Nikon E600 microscope).
Histological examination of bone marrow
Tibias collected from animals with and without BMT were fixed in PBS-buffered formalin
(4%), decalcified in Osteosoft (Merck, Germany), paraffin-embedded, sectioned along the
mid-sagittal plane in 3 μm-sections. Sections were stained with haematoxylin and eosin.
Immunofluorescence for eNOS was performed as described above to exclude the effect of
eNOS deficiency in the recipient on the engraftment of eNOS-positive bone marrow. eNOSpositive and negative cells were counted in 5 randomly chosen microscopic fields. The
percentage of eNOS-positive cells in eNOS-/- / WT-mice 4 and 9 weeks after bone-marrow
transplantation varied between 80-90% of the non-transplanted wild-type mice.
Supplementary Figure Legends
Fig. 6 Morphology of the bone marrow in bone-marrow transplanted mice
(A) shows representative examples of bone marrow sections of bone-marrow transplanted
animals 4 weeks after bone-marrow transplantation and control animals without BMT stained
with haematoxylin and eosin. There is no significant morphological difference between bonemarrow transplanted animals and corresponding controls. Bars = 30µm
(B) depicts eNOS-positive cells in the bone marrow of an eNOS-/- BMT mouse received
wildtype bone marrow, eNOS (red). Nuclei are stained blue by DAPI. eNOS-positive cells are
marked by arrows. Bars = 10µm.
2