Mice were perfused with 4% paraformaldehyde in phosphate-buffered saline
(PBS). After perfusion, brain blocks with SVZ were removed and
postfixed in 4% paraformaldehyde at 4°C for 24 hr. The blocks were
then equilibrated in sucrose (30% in PBS) and embedded in optimal
cutting temperature compound and kept frozen at −80°C. Cryosections
were cut at a thickness of 30 μm. Sections were incubated in 1N HCl at
0°C for 5 min and 2N HCl at 37°C for 15 min and washed with PBS before
immunohistochemical staining. Sections were then blocked in 3% normal
donkey serum in PBS with 1% Triton X-100 for 30 min and further
incubated in the following primary antibodies 4°C overnight: BrdU
(mouse, 1:200, Developmental Studies Hybridoma Bank, G3G4), SOX2
(rabbit, 1:200, Cell Signaling Technology, #2748), doublecortin (DCX;
goat,1:200, Santa Cruz Biotechnology, C-18). After three washes with
PBS, the samples were incubated in an appropriate secondary antibodies
coupled to Alexa488 or Alexa 568 (Invitrogen) at a dilution of 1:1,000
at room temperature for 2 hr. After washing with PBS for 3 times, the
sections were mounted with Vectashield mountain medium with DAPI
(Vector Laboratory).
(PBS). After perfusion, brain blocks with SVZ were removed and
postfixed in 4% paraformaldehyde at 4°C for 24 hr. The blocks were
then equilibrated in sucrose (30% in PBS) and embedded in optimal
cutting temperature compound and kept frozen at −80°C. Cryosections
were cut at a thickness of 30 μm. Sections were incubated in 1N HCl at
0°C for 5 min and 2N HCl at 37°C for 15 min and washed with PBS before
immunohistochemical staining. Sections were then blocked in 3% normal
donkey serum in PBS with 1% Triton X-100 for 30 min and further
incubated in the following primary antibodies 4°C overnight: BrdU
(mouse, 1:200, Developmental Studies Hybridoma Bank, G3G4), SOX2
(rabbit, 1:200, Cell Signaling Technology, #2748), doublecortin (DCX;
goat,1:200, Santa Cruz Biotechnology, C-18). After three washes with
PBS, the samples were incubated in an appropriate secondary antibodies
coupled to Alexa488 or Alexa 568 (Invitrogen) at a dilution of 1:1,000
at room temperature for 2 hr. After washing with PBS for 3 times, the
sections were mounted with Vectashield mountain medium with DAPI
(Vector Laboratory).