Biology Reference
In-Depth Information
immunohistochemical staining were observed by two pathologists in a blinded
manner.
Fluorescent in Situ Hybridization (FISH)
Fluorescent in situ hybridization was performed according to the mouse Y chro-
mosome probe manual (STAR*FISH; Cambio, Cambridge, England) with some
modifications. Cryostat sections of 5
µ
m in thickness were fixed in Carnoy's fixa-
tive for three times, 10 min each, and the sections were incubated in pepsin solu-
tion (1% in 0.1N HCl) for 10 minutes and rinsed in PBS. Serial ethanol dehydra-
tion was done (1.5 min each), and the slides were air-dried at room temperature.
Sections were denatured at 65°C for 2 min in preheated 70% formamide and
2×SSC buffer, pH 7.0, and were then 'quenched' with ice-cold 70% ethanol for
1.5 min. Serial ethanol dehydration was done again. The mouse Y chromosome
probe labeled with Cy3 was denatured at 65°C for 10 min and applied to the sec-
tions. The sections were coverslipped and sealed with rubber cement for incuba-
tion overnight in a hydrated slide box at 37°C. The next day, the coverslips were
carefully removed. The sections were washed twice in preheated 50% formamide
in 2×SSC buffer for 5 min each at 45°C and were then gently washed twice in
preheated 1×SSC buffer for 5 min each at 45°C.
statistical Assay
Significance was determined using one-way ANOVA and log-rank test (SPSS 11.0
for windows). Difference between groups were significant at a value of P < 0.05.
results
Bmscs Isolation, Genetic Modification and Sflt-1 Function
Validation
After harvest of monolayer cells with spindle-like morphology from mouse bone
marrow, three CD markers (CD34, CD29 and CD44) were used for character-
izing BMSCs. The cells were positive for CD29, CD44, and negative for CD34,
indicating that they didn't belong to hematopoietic progenitor cells, but rather
bone marrow-derived stromal cells (BMSCs) or mesenchymal stem cells (MSCs).
BMSCs were cultured to reach to 90% confluence and incubated with adenovi-
ruses at a MOI of 3000 for 2 hours. Twenty four hours later, all of the cells were
GFP-positive checked by fluorescence microscopy and the cells were ready for tail
vein infusion after PBS washing two times (Figure 1A). The secreted sFlt-1 in the
Search WWH ::
Custom Search