The plates were first rinsed once
in PBS and then fixed in 10% formaldehyde for 20 min. The formaldehyde
was aspirated, and the plates were washed 3 times, 5 min each, in
PBS/0.1% Triton-X 100. The plates were then incubated with respective
primary antibodies in PBS/0.1% Triton-X 100/1% BSA overnight at 4
°C. The samples were then washed 6 times, 5 min each, in PBS/0.1%
Triton-X 100. Secondary antibodies (1:500) in PBS/0.1% Triton-X 100/1%
BSA were incubated with the samples at room temperature for 1 h. The
samples were then washed 3 times in PBS and mounted with Vectashield
containing DAPI. Fluorescent images were acquired using a Zeiss Axioskop2
coupled to an MRm (Zeiss) camera at 10×, 20×, and 40×
magnifications with Axiovision software. The primary and secondary
antibody sources and dilutions were as follows: mouse anti-nestin
from BD Biosciences Pharmingen (1:500), mouse monoclonal antineuronal
class III β-tubulin (TuJ1) from Nordic Biosite (1:500), mouse
monoclonal anti-a-smooth muscle actin (SMA) from Sigma (1:1000), rabbit
polyclonal anti-CD133 from Invitrogen (1:500), and rabbit polyclonal
antiglial fibrillary acidic protein (GFAP) from DAKO (1:500). Species-specific
Alexa-488 and Alexa-594-conjugated secondary antibodies were used
as appropriate and were obtained from Molecular Probes (1:500).
in PBS and then fixed in 10% formaldehyde for 20 min. The formaldehyde
was aspirated, and the plates were washed 3 times, 5 min each, in
PBS/0.1% Triton-X 100. The plates were then incubated with respective
primary antibodies in PBS/0.1% Triton-X 100/1% BSA overnight at 4
°C. The samples were then washed 6 times, 5 min each, in PBS/0.1%
Triton-X 100. Secondary antibodies (1:500) in PBS/0.1% Triton-X 100/1%
BSA were incubated with the samples at room temperature for 1 h. The
samples were then washed 3 times in PBS and mounted with Vectashield
containing DAPI. Fluorescent images were acquired using a Zeiss Axioskop2
coupled to an MRm (Zeiss) camera at 10×, 20×, and 40×
magnifications with Axiovision software. The primary and secondary
antibody sources and dilutions were as follows: mouse anti-nestin
from BD Biosciences Pharmingen (1:500), mouse monoclonal antineuronal
class III β-tubulin (TuJ1) from Nordic Biosite (1:500), mouse
monoclonal anti-a-smooth muscle actin (SMA) from Sigma (1:1000), rabbit
polyclonal anti-CD133 from Invitrogen (1:500), and rabbit polyclonal
antiglial fibrillary acidic protein (GFAP) from DAKO (1:500). Species-specific
Alexa-488 and Alexa-594-conjugated secondary antibodies were used
as appropriate and were obtained from Molecular Probes (1:500).