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

Tsuji et. al. amniotic membrane-derived stem cell
(2009/205260-R3)
PFA (4ºC) for 20 minutes, and rinsed with phosphate-buffered saline
(PBS) three times. Then cells were fixed in 0.02% triton-X 200 for 20
minutes at room temperature, and rinsed with PBS. Cells were incubated
with primary antibodies: anti-albumin (1:1000; abcam ab8940), antiglucagon
(prediluted; abcam ab930), anti-glial fibrillary acidic protein
(GFAP) (1:200; Santa Cruz Biotechnology sc9065), anti-nestin (1:200;
abcam ab22035), anti-osteocalcin (1:50; Acris BP710), or anti-collagen
type-II (1:20; Cosmobio MNS-PS042) for 1 hour at room temperature.
Dilution buffer without primary antibody was used as the negative control.
The samples were rinsed with PBS three times and incubated with FITCconjugated
secondary antibodies for 30 minutes at room temperature.
Cells were rinsed with PBS and mounted with fluorescent mounting
medium (Dako Cytomation S3023).
4. Calculation of number of surviving EGFP-positive cardiomyocytes in vivo
Immediately after the hearts were excised, they were fixed by 4%
paraformaldehyde (PFA) for 2 days at 4ºC. Then tissue was dehydrated
by 10% sucrose containing PBS for 6 hours and then 20 % sucrose
containing PBS for 6 hours. The dehydrated tissue was dipped with
optimal cutting temperature (OCT) compound, and then the tissue was
quickly frozen by liquid nitrogen. The whole tissue specimens were
sliced by the cryostat (6 µm thickness), at intervals of 360 µm, then
completely dried under air flow for 2 hours. They were fixed again by 4%
PFA for 30 minutes, subsequently treated by 0.2% triton-X containing
PBS solution for 30 minutes, and then immunohistochemistry was
performed. Stained samples were observed by fluorescence microscope
(IX-71, Olympus, Tokyo, JAPAN) with 10x objective lens
(UPLFLN10XPH). EGFP-positive cardiomyocytes were defined by cells
having clear striation staining pattern of cardiac troponin-I. The number
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