Tsuji et. al. amniotic membrane-derived stem cell (2009/205260-R3 ...

Tsuji et. al. amniotic membrane-derived stem cell
(2009/205260-R3)
each heart was sliced every 50 µm or 100 µm. Hematoxylin-eosin
staining was performed for a slice in which EGFP-positive clusters of
cells were observed.
7. Immunohistochemical analysis for angiogenesis in vivo
Paraffin-embedded heart tissues were sectioned every 4µm, then
myocardial infarction areas were selected. After deparaffinization and
antigen retrieval, sample was pretreated with 5% skim milk in Trisbuffered
saline (TBS) followed by anti-rat CD34 antibody (1:200, R&D
Systems: AF4117) at 4º C for over night. Next day, each slide was
washed with TBS and applied with biotinylated Goat immunoglobulins
(Dako; E0466), next with strept ABC complex/HRP (Dako; K0377) and
then with 3,3`-Diaminobenzine (DAB) substrate (Wako: K3183500).
Finally, each sample was counterstained with Hematoxylin. Each
sample was observed by light microscope (x100) and 5 digital images
from the normal myocardium and the center of the myocardial infarction
zone were collected at random. Images were randomized and analyzed
by the blinded observer. % brown pixel area in the whole heart tissue
was calculated by Photoshop (Adobe). The data was averaged and
shown in Online. Fig XI.
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