【求助】大鼠干细胞如何鉴定？
这个帖子发布于 19 年零 306 天前，其中的信息可能已发生改变或有所发展。
大鼠干细胞如何鉴定？我做肌源性干细胞（MDSC），如何鉴定是干细胞？可以 用CD34 Ab吗？可是没找到针对大鼠的抗体呀？有篇文献写的是用抗人
CD34抗体，不明白。请大侠指教？
CML contents of CML-HSA prepared were 18.01 moles
CML per mole HSA.
Preparation of Choroidal Explant Choroidal explants
of deeply anesthetized normal Wistar rats were prepared as
previously reported.6,31) Blood vessels, connective and fatty
tissues in the outside of sclera in eye balls of these rats were
removed in Dulbecco’s modified Eagle’s medium (DMEM,
Nissui, Tokyo) containing 10% heat-inactive fetal bovine
serum (FBS, JRH Bioscience, Lenexa, KS, U.S.A.),
160 U/ml benzylpenicillin potassium (Banyu Seiyaku,
Tokyo) and 0.1 mg/ml streptomycin sulfate (Meiji Seika,
Tokyo). After removal of the cornea, lens, corpus vitreum
and retina from the eye balls, similar size of choroidal explants
were carefully isolated with forceps from the interior
aspect of the sclera in 10% FBS-DMEM in the presence of
antibiotics, using a surgical microscope. The size of isolated
explant was approximately 0.16mm2. In some experiments,
the same shape and size (1.8mm2) of choroidal explants connected
with sclera was isolated using ophthalmic surgical instrument.
Culture of Choroidal Explants The choroidal explants
were cultured as reported previously.6,7,31) The similar size of
explants were plated on fibrin gels prepared by mixing 3mg
fibrinogen (0.3 ml, Sigma), and 1U thrombin (Sigma) per ml
DMEM containing antibiotics in 16-mm dish (Corning,
Corning, NY, U.S.A.). The same volumes of a mixture of the
above concentrations of fibrinogen and thrombin solutions
were overlaid and solidified. The choroidal explants were
cultured with 5% FBS- or 1% FBS-DMEM (0.5 ml) containing
antibiotics and 300 mg/ml e-amino caproic acid in the
presence or absence of CML at 37 °C under 5% CO2 and
95% air. Media were changed every other day. In some experiments,
cells composed in vessel-like structures were isolated
with 0.75% collagenase (Wako, Osaka)-PBS, washed
with PBS and plated on collagen-coated dish for 24 h.
Light and Electron Microscopy and Immunostaining
The explants fixed in 10% formalin were processed for paraf-
fin-embedded histological sections. They were stained with
hematoxylin and eosin. The explants for transmission electron
microscopy were fixed in 1% glutaraldehyde, postfixed
in 1% osmium tetroxide, embedded in an epoxy resin, ultrathin-
cut and stained with uranium and lead citrate. The explants
for immunostaining were fixed in 4% paraformaldehyde,
embedded in paraffin and incubated with antibody
against human CD34 (H-140, sc-9095m Cosmo Bio Co.,
Ltd., Tokyo), a marker of vascular endothelial cells and their
progenitor cells.32) The negative controls were incubated with
non-immune rabbit IgG (Dako Japan, Kyoto). 3Y1 cells, a
cloned rat fibroblast33) (Riken Cell Bank, Tsukuba) cultured
in fibrin gel were also used for a negative control. Immunostained
cells were processed using LSAB2 kit for rat with
horseradish peroxidase (HRP) and 3,3-diaminobenzidine
tetrahydrochloride (DAB) (Dako Japan).
Assessment of Neovascularization Vessel-like structures
newly budded from cultured explants of choroidal tissues
were photographed with an Olympus camera equipped
with a CKS microscope (Olympus, Tokyo) or Polaroid
PDMC II/OL with a CK40 microscope (Olympus). Typical
photograph of vessel-like structures in cultured choroidal explant
was shown in Fig. 1. The number of budded structures
per explant was counted under 40 magnification and used
as an index of in vitro choroidal neovascularization.6)
Statistical Analysis All values were expressed as
meansS.E.M. Differences between group data were evaluated
by one-way analysis of variance followed by the multiple
range test of Scheffé at p0.05 or 0.01. A value of
p0.05 was considered statistically significant.
RESULTS
Results of Light and Electron Microscopy Choroidal
explant was cultured in fibrin gel for 6 d in DMEM with 5%
FBS. Vessel-like structures were budded from 2 d in culture
and developed from choroidal explants in a time-dependent
manner. The cytoplasmic processes radiating from the
choroidal explants were variable in length and mostly less
than 20 mm in outer diameter. They had long ellipsoid nuclei
and long and thin cytoplasmic processes. Histological sections
cut along their long axis showed occasionally lumina
surrounded by attenuated cellular extensions (Figs. 2①, ②,
asterisks). However, the lumina were too narrow to allow
passage of erythrocytes. In transmission electron microscopy,
the attenuated cell processes had numerous membranebound,
polymorphic vesicles (Figs. 2③, ④, arrows). They
were present everywhere in the cell, not only in the cell extensions
but also in the center of cells. They included flocculent
material with a low electron density similar to the background
substance of the connective tissue. Extensive survey
of the vesicle, however, failed to find the presence of collagen
fibers in the vesicles. The vesicles were often opened to
CD34抗体，不明白。请大侠指教？
CML contents of CML-HSA prepared were 18.01 moles
CML per mole HSA.
Preparation of Choroidal Explant Choroidal explants
of deeply anesthetized normal Wistar rats were prepared as
previously reported.6,31) Blood vessels, connective and fatty
tissues in the outside of sclera in eye balls of these rats were
removed in Dulbecco’s modified Eagle’s medium (DMEM,
Nissui, Tokyo) containing 10% heat-inactive fetal bovine
serum (FBS, JRH Bioscience, Lenexa, KS, U.S.A.),
160 U/ml benzylpenicillin potassium (Banyu Seiyaku,
Tokyo) and 0.1 mg/ml streptomycin sulfate (Meiji Seika,
Tokyo). After removal of the cornea, lens, corpus vitreum
and retina from the eye balls, similar size of choroidal explants
were carefully isolated with forceps from the interior
aspect of the sclera in 10% FBS-DMEM in the presence of
antibiotics, using a surgical microscope. The size of isolated
explant was approximately 0.16mm2. In some experiments,
the same shape and size (1.8mm2) of choroidal explants connected
with sclera was isolated using ophthalmic surgical instrument.
Culture of Choroidal Explants The choroidal explants
were cultured as reported previously.6,7,31) The similar size of
explants were plated on fibrin gels prepared by mixing 3mg
fibrinogen (0.3 ml, Sigma), and 1U thrombin (Sigma) per ml
DMEM containing antibiotics in 16-mm dish (Corning,
Corning, NY, U.S.A.). The same volumes of a mixture of the
above concentrations of fibrinogen and thrombin solutions
were overlaid and solidified. The choroidal explants were
cultured with 5% FBS- or 1% FBS-DMEM (0.5 ml) containing
antibiotics and 300 mg/ml e-amino caproic acid in the
presence or absence of CML at 37 °C under 5% CO2 and
95% air. Media were changed every other day. In some experiments,
cells composed in vessel-like structures were isolated
with 0.75% collagenase (Wako, Osaka)-PBS, washed
with PBS and plated on collagen-coated dish for 24 h.
Light and Electron Microscopy and Immunostaining
The explants fixed in 10% formalin were processed for paraf-
fin-embedded histological sections. They were stained with
hematoxylin and eosin. The explants for transmission electron
microscopy were fixed in 1% glutaraldehyde, postfixed
in 1% osmium tetroxide, embedded in an epoxy resin, ultrathin-
cut and stained with uranium and lead citrate. The explants
for immunostaining were fixed in 4% paraformaldehyde,
embedded in paraffin and incubated with antibody
against human CD34 (H-140, sc-9095m Cosmo Bio Co.,
Ltd., Tokyo), a marker of vascular endothelial cells and their
progenitor cells.32) The negative controls were incubated with
non-immune rabbit IgG (Dako Japan, Kyoto). 3Y1 cells, a
cloned rat fibroblast33) (Riken Cell Bank, Tsukuba) cultured
in fibrin gel were also used for a negative control. Immunostained
cells were processed using LSAB2 kit for rat with
horseradish peroxidase (HRP) and 3,3-diaminobenzidine
tetrahydrochloride (DAB) (Dako Japan).
Assessment of Neovascularization Vessel-like structures
newly budded from cultured explants of choroidal tissues
were photographed with an Olympus camera equipped
with a CKS microscope (Olympus, Tokyo) or Polaroid
PDMC II/OL with a CK40 microscope (Olympus). Typical
photograph of vessel-like structures in cultured choroidal explant
was shown in Fig. 1. The number of budded structures
per explant was counted under 40 magnification and used
as an index of in vitro choroidal neovascularization.6)
Statistical Analysis All values were expressed as
meansS.E.M. Differences between group data were evaluated
by one-way analysis of variance followed by the multiple
range test of Scheffé at p0.05 or 0.01. A value of
p0.05 was considered statistically significant.
RESULTS
Results of Light and Electron Microscopy Choroidal
explant was cultured in fibrin gel for 6 d in DMEM with 5%
FBS. Vessel-like structures were budded from 2 d in culture
and developed from choroidal explants in a time-dependent
manner. The cytoplasmic processes radiating from the
choroidal explants were variable in length and mostly less
than 20 mm in outer diameter. They had long ellipsoid nuclei
and long and thin cytoplasmic processes. Histological sections
cut along their long axis showed occasionally lumina
surrounded by attenuated cellular extensions (Figs. 2①, ②,
asterisks). However, the lumina were too narrow to allow
passage of erythrocytes. In transmission electron microscopy,
the attenuated cell processes had numerous membranebound,
polymorphic vesicles (Figs. 2③, ④, arrows). They
were present everywhere in the cell, not only in the cell extensions
but also in the center of cells. They included flocculent
material with a low electron density similar to the background
substance of the connective tissue. Extensive survey
of the vesicle, however, failed to find the presence of collagen
fibers in the vesicles. The vesicles were often opened to
