Rabbit anti satb2

For immunofluorescent staining, sections were rinsed in PBS and blocked with 5% normal serum/0.1% Triton X-100/PBS at room temperature. Primary antibodies were diluted in blocking solution and incubated 1–2 days at 4°C with gentle agitation. The antibodies utilized were; rabbit anti-Parvalbumin (Swant), chicken anti-EGFP (Aves Labs), rat anti-CTIP2 (Abcam), rabbit anti-SATB2 (Abcam), rabbit anti-CUX1 (Santa Cruz), mouse anti-NEUN (Chemicon), rabbit anti-Cleaved Caspase-3 (Cell Signaling Technology), rabbit anti-IBA1 (Wako), goat anti-IGF1 (R&D Research), rabbit anti-PKCγ (Santa Cruz), rabbit anti-MAP2K1/2(MEK1/2) (Abcam), rabbit anti-P-MAPK1/3(ERK1/2) (Cell Signaling Technology) and rabbit anti-IGF1Rβ (Cell signaling Technology). After rinsing in PBS/T, the secondary antibody was diluted in blocking solution and added overnight at 4°C. Secondary antibodies included Alexa Fluor 488, 546 or 568, and 647 conjugated anti-rabbit, anti-mouse, anti-rat, or anti-goat IgG (Invitrogen). For some experiments slides were then incubated in Hoechst or DAPI for nuclear labeling, rinsed, and mounted. Images were collected with a Zeiss LSM 710, 780, or Leica SP5 laser scanning confocal microscope.

Mice were perfused with 4% paraformaldehyde (PFA) at different postnatal ages. All brains were fixed in 4% PFA overnight, cryoprotected in 30% sucrose in phosphate-buffered saline overnight and cut into 20 μm-thick sections. For immunohistochemistry, brain sections were incubated with rabbit anti-Satb2 (1:300, Abcam) or goat anti-5-HTT antibody (1:1000, Immunostar) at 4°C overnight, and then incubated with biotinylated horse anti-rabbit IgG or horse anti-goat IgG (1:500, Jackson ImmunoResearch) at room temperature for 3 h followed by incubation with streptavidin-Cy3 (1:1000, Jackson ImmunoResearch) and counterstaining with Hoechst 33258 (1:2000, Sigma) at room temperature for 1 h.
The AuCl₃ staining was performed as a previous study (Wahlsten et al., 2003 (link)). The brain sections were stained with 0.2% gold chloride (AuCl₃) in phosphate buffer. The process was taken place in darkness. Once axonal staining became evident, the reaction was stopped by transferring sections to 2.5% sodium thiosulfate anhydrous for 5 min.
The antisense digoxigenin-labeled RNA probes of RORβ, Cux2, Ctip2, and Tle4 were synthesized according to the Allen Brain Atlas website, and in situ hybridization was performed as described in our previous study (Song et al., 2011 (link)).