Immunohistochemical Profiling of Embryonic and Postnatal Brain

Immunohistochemistry was performed as previously described [26 ]. Briefly, postnatal and embryonic brains were collected and placed in 4% paraformaldehyde overnight at 4 °C, cryoprotected in 30% sucrose for at least 24 h, frozen in optimal cutting temperature and cryosectioned. All tissues were sectioned coronally at 12 or 20 μm and stained on glass slides.
For SP9, BCL11B, and SIX3 immunohistochemistry, sections were boiled briefly in 10 mmol/L sodium citrate for antigen retrieval. Immunohistochemistry for BrdU⁺ cells was performed after 45 min of incubation in 2 N HCl and rinsing twice in 0.1 mol/L borate buffer at room temperature. Immunofluorescence labeling was performed with the following primary antibodies: rat anti-BCL11B (Abcam, Ab18465), rat anti-BrdU (Accurate Chemical, OBT0030s), chicken anti-β-gal (Abcam, ab9361), rabbit anti-cleaved Caspase-3 (Cell Signaling, #9661), rabbit anti-CRE (Millipore, 69050-3), rabbit anti-EBF1 (Merck, AB10523), rabbit anti-FOXP1 (Abcam, Ab16645), rabbit anti-KI67 (Abcam, ab15580), mouse anti-SIX3 (Santa Cruz Biotechnology, sc-398797), goat anti-SP8 (Santa Cruz Biotechnology, sc-104661), rabbit anti-SP9 [25 (link)]. Appropriate Alexa Fluor 488-, Cy3- or Alexa Fluor 647-conjugated secondary antibodies from Jackson ImmunoResearch were used.